Soybean transcription terminators and use in expression of transgenic genes in plants

ABSTRACT

Five novel plant transcription terminators MYB2, KTI1, PIP1, EF1A2, and MTH1 are isolated from soybean and their functions in the regulation of RNA transcription and processing in plants are described.

This application is a Divisional of U.S. application Ser. No.13/288,992, filed Nov. 4, 2011, now Allowed. U.S. application Ser. No.13/288,992 is a Divisional of U.S. application Ser. No. 12/486,793 filedJun. 18, 2009, now U.S. Pat. No. 8,080,413, granted Dec. 20, 2011. U.S.application Ser. No. 12/486,793 claims the benefit of U.S. ProvisionalApplication No. 61/073,389, filed Jun. 18, 2008, the entire content ofwhich is herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to novel plant transcription terminators MYB2,KTI1, PIP1, EF1A2, and MTH1 isolated from soybean and their use in theregulation of gene expression in plants.

BACKGROUND OF THE INVENTION

Recent advances in plant genetic engineering have opened new doors toengineer plants with improved characteristics or traits, such as plantdisease resistance, insect resistance, drought tolerance, extremetemperature tolerance, herbicidal resistance, yield improvement,improvement of the nutritional quality of the edible portions of theplant, and enhanced stability or shelf-life of the ultimate consumerproduct obtained from the plants. Thus, a desired gene (or genes) withthe molecular function to impart different or improved characteristicsor qualities, can be incorporated into a plant's genome. The newlyintegrated gene (or genes) coding sequence can then be expressed in theplant cell to exhibit the desired new trait or characteristic. It isimportant that appropriate regulatory signals be present in properconfigurations in order to obtain expression of the newly inserted genecoding sequence in the plant cell. These regulatory signals typicallyinclude a promoter region, a 5′ non-translated leader sequence, and a 3′transcription termination/polyadenylation sequence.

A promoter is a non-coding genomic DNA sequence, usually upstream (5′)to the relevant coding sequence, to which RNA polymerase binds beforeinitiating transcription. This binding aligns the RNA polymerase so thattranscription will initiate at a specific transcription initiation site.The insertion of promoter sequences in recombinant DNA constructsdictates when and where in the plant the introduced DNA sequences willbe expressed.

In contrast, sequences located downstream (3′) to the relevant codingsequence, i.e. transcription terminators, appear to control quantitativelevels of expression (Ali and Taylor, Plant Mol. Biol. 46:251-61(2001)). Transcription terminators function to stop transcription andalso have important effects on the processing and degradation of RNAstrands generated by transcription. In recombinant DNA constructs,terminators are typically inserted immediately after the 3′-end of thetranslated region of a gene of interest.

Recombinant DNA constructs may contain more than one gene cassette, eachconsisting of a promoter, gene of interest, and a terminator. If RNAtranscription is not terminated effectively, the transcription of onegene cassette may interfere with the expression of a gene in anothercassette. Similarly, unwanted transcription of trait-unrelated(downstream) sequences may interfere with trait performance. Weakterminators, for example, can generate read-through, thereby affectingthe expression of genes located in neighboring expression cassettes(Padidam and Cao, Biotechniques 31:328-30, 332-4 (2001)). However, theuse of appropriate transcription terminators in recombinant DNAconstructs can minimize read-through into downstream sequences (e.g.,other expression cassettes) and allow more efficient recycling of RNApolymerase II, thereby improving gene expression.

Often, the same transcription termination sequence is used multipletimes in one transgenic organism, sometimes resulting in unintendedsilencing. Thus, there is a demand for alternative transcriptiontermination sequences. Unfortunately, the prediction of functional,efficient transcription termination sequences by bioinformatics isdifficult since virtually no conserved sequences exist to allow for sucha prediction. Thus, there is an ongoing interest in the isolation ofnovel terminators that are capable of controlling transcriptiontermination and that improve gene expression.

SUMMARY OF THE INVENTION

In a first embodiment, this invention concerns a terminator, whereinsaid terminator comprises the nucleotide sequence set forth in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124,SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, or SEQ ID NO:128; afull-length complement of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ IDNO:127, or SEQ ID NO:128; or a nucleotide sequence having at least 90%sequence identity, based on the BLASTN method of alignment, whencompared to the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ IDNO:126, SEQ ID NO:127, or SEQ ID NO:128, or a full-length complementthereof.

In a second embodiment, the invention concerns a terminator, whereinsaid terminator is a nucleotide sequence comprising a fragment of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124,SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, or SEQ ID NO:128; afull-length complement of a fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ IDNO:126, SEQ ID NO:127, or SEQ ID NO:128; or a nucleotide sequence havingat least 90% sequence identity, based on the BLASTN method of alignment,when compared to the fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, or SEQ ID NO:128, or a full-length complement thereof.

In a third embodiment, this invention concerns a recombinant DNAconstruct comprising a promoter, at least one heterologous nucleic acidfragment, and the terminator of the invention, wherein the promoter,heterologous nucleic acid fragment, and terminator are operably linked.

In a fourth embodiment, this invention concerns a vector, cell, plant,or seed comprising a recombinant DNA construct of the presentdisclosure.

In a fifth embodiment, this invention concerns plants comprising thisrecombinant DNA construct and seeds obtained from such plants.

In a sixth embodiment, this invention concerns a method of expressing atleast one heterologous nucleic acid fragment in a plant cell whichcomprises:

-   -   (a) transforming a plant cell with the recombinant DNA construct        described above;    -   (b) growing fertile mature plants from the transformed plant        cell of step (a);    -   (c) selecting plants containing the transformed plant cell        wherein the heterologous nucleic acid fragment is expressed.

In a seventh embodiment, this invention concerns a method of altering amarketable plant trait. The marketable plant trait concerns genes andproteins involved in disease resistance, herbicide resistance, insectresistance, carbohydrate metabolism, fatty acid metabolism, amino acidmetabolism, plant development, plant growth regulation, yieldimprovement, drought resistance, cold resistance, heat resistance, andsalt resistance.

BRIEF DESCRIPTION OF SEQUENCES AND DRAWINGS

The invention can be more fully understood from the following detaileddescriptions, the drawings, and the sequence descriptions that form apart of this application. The Sequence Listing contains the one lettercode for nucleotide sequence characters and the three letter codes foramino acids as defined in conformity with the IUPAC-IUBMB standardsdescribed in Nucleic Acids Research 13:3021-3030 (1985) and in theBiochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

SEQ ID NO:1 is the nucleotide sequence of the soybean MYB2 terminatorcloned into DNA construct QC339.

SEQ ID NO:2 is the nucleotide sequence of the soybean KTI1 terminatorcloned into DNA construct QC340.

SEQ ID NO:3 is the nucleotide sequence of the soybean PIP1 terminatorcloned into DNA construct QC350.

SEQ ID NO:4 is the nucleotide sequence of the soybean EF1A2 terminatorcloned into DNA construct QC351.

SEQ ID NO:5 is the nucleotide sequence of the soybean MTH1 terminatorcloned into DNA construct QC352.

SEQ ID NO:6 is the 1061 bp nucleotide sequence of the putative soybeantranscription factor MYB2 gene PSO323364. Nucleotides 1 to 144 represent5′ untranslated sequence, while the coding sequence is found atnucleotides 145 to 756, with the translation initiation codon atnucleotides 145 to 147 and the termination codon at nucleotides 757 to759. Nucleotides 757 to 1061 are part of the 3′ untranslated sequence.

SEQ ID NO:7 is the 882 bp nucleotide sequence of the putative soybeanKunitz trypsin inhibitor KTI1 gene PSO400362. Nucleotides 1 to 30represent 5′ untranslated sequence, while the coding sequence is foundat nucleotides 31 to 639, with the translation initiation codon atnucleotides 31 to 33 and the termination codon at nucleotides 640 to642. Nucleotides 640 to 882 are part of the 3′ untranslated sequence.

SEQ ID NO:8 is the 1247 bp nucleotide sequence of the putative soybeanaquaporin protein PIP1 gene PSO332986. Nucleotides 1 to 67 represent 5′untranslated sequence, while the coding sequence is found at nucleotides68 to 934, with the translation initiation codon at nucleotides 68 to 70and the termination codon at nucleotides 935 to 937. Nucleotides 935 to1247 are part of the 3′ untranslated sequence.

SEQ ID NO:9 is the 1772 bp nucleotide sequence of the putative soybeantranslation elongation factor EF1 alpha homolog (EF1A2) gene PSO333268.Nucleotides 1 to 86 represent 5′ untranslated sequence, while the codingsequence is found at nucleotides 87 to 1427, with the translationinitiation codon at nucleotides 87 to 89 and the termination codon atnucleotides 1428 to 1430. Nucleotides 1428 to 1772 are part of the 3′untranslated sequence.

SEQ ID NO:10 is the 574 bp nucleotide sequence of the putative soybeantype 2 metallothionein MTH1 gene PSO333209. Nucleotides 1 to 78represent 5′ untranslated sequence, while the coding sequence is foundat nucleotides 79 to 315, with the translation initiation codon atnucleotides 79 to 81 and the termination codon at nucleotides 316 to318. Nucleotides 316 to 574 are part of the 3′ untranslated sequence.

SEQ ID NO:11 is the predicted amino acid sequence of the protein encodedby the putative soybean transcription factor MYB2 gene PSO323364 (SEQ IDNO:6).

SEQ ID NO:12 is the predicted amino acid sequence of the protein encodedby the putative soybean Kunitz trypsin inhibitor KTI1 gene PSO400362(SEQ ID NO:7).

SEQ ID NO:13 is the predicted amino acid sequence of the protein encodedby the putative soybean aquaporin protein PIP1 gene PSO332986 (SEQ IDNO:8).

SEQ ID NO:14 is the predicted amino acid sequence of the protein encodedby the putative soybean translation elongation factor EF1 alpha homolog(EF1A2) gene PSO333268 (SEQ ID NO:9).

SEQ ID NO:15 is the predicted amino acid sequence of the protein encodedby the putative soybean type 2 metallothionein MTH1 gene PSO333209 (SEQID NO:10).

SEQ ID NO:16 is the MPSS tag sequence specific to the PSO323364 gene.

SEQ ID NO:17 is the MPSS tag sequence specific to the PSO400362 gene.

SEQ ID NO:18 is the MPSS tag sequence specific to the PSO332986 gene.

SEQ ID NO:19 is the MPSS tag sequence specific to the PSO333268 gene.

SEQ ID NO:20 is the sense primer ATPS-87F for qRT-PCR of the endogenouscontrol gene ATP sulfurylase (ATPS).

SEQ ID NO:21 is the antisense primer ATPS-161R for qRT-PCR of theendogenous control gene ATPS.

SEQ ID NO:22 is the sense primer PSO323364F for qRT-PCR analysis of thePSO323364 gene.

SEQ ID NO:23 is the antisense primer PSO323364R for qRT-PCR analysis ofthe PSO323364 gene.

SEQ ID NO:24 is the sense primer PSO400362F for qRT-PCR analysis of thePSO400362 gene.

SEQ ID NO:25 is the antisense primer PSO400362R for qRT-PCR analysis ofthe PSO400362 gene.

SEQ ID NO:26 is the sense primer PSO332986F for qRT-PCR analysis of thePSO332986 gene.

SEQ ID NO:27 is the antisense primer PSO332986R for qRT-PCR analysis ofthe PSO332986 gene.

SEQ ID NO:28 is the sense primer PSO333268F for qRT-PCR analysis of thePSO333268 gene.

SEQ ID NO:29 is the antisense primer PSO333268R for qRT-PCR analysis ofthe PSO333268 gene.

SEQ ID NO:30 is the sense primer PSO333209F for qRT-PCR analysis of thePSO333209 gene.

SEQ ID NO:31 is the antisense primer PSO333209R for qRT-PCR analysis ofthe PSO333209 gene.

SEQ ID NO:32 is the 5232 bp sequence of the DNA construct QC315.

SEQ ID NO:33 is the 5492 bp sequence of the DNA construct QC327.

SEQ ID NO:34 is the 8409 bp sequence of the DNA construct QC324i.

SEQ ID NO:35 is the 10017 bp sequence of the DNA construct QC339.

SEQ ID NO:36 is the 10031 bp sequence of the DNA construct QC340.

SEQ ID NO:37 is the 9995 bp sequence of the DNA construct QC350.

SEQ ID NO:38 is the 9922 bp sequence of the DNA construct QC351.

SEQ ID NO:39 is the 9939 bp sequence of the DNA construct QC352.

SEQ ID NO:40 is the oligonucleotide primer PSO323364Sac used as a senseprimer in the PCR amplification of the MYB2 terminator (PSO323364) fromthe soybean genome. A SacI recognition site (GAGCTC) was added forsubsequent cloning.

SEQ ID NO:41 is the oligonucleotide primer PSO323364Eco used as anantisense primer in the PCR amplification of the MYB2 terminator(PSO323364) from the soybean genome. An EcoRI recognition site (GAATTC)was added for subsequent cloning.

SEQ ID NO:42 is the oligonucleotide primer PSO400362Sac used as a senseprimer in the PCR amplification of the KTI1 terminator (PSO400362) fromthe soybean genome. A SacI recognition site (GAGCTC) was added forsubsequent cloning.

SEQ ID NO:43 is the oligonucleotide primer PSO400362Eco used as anantisense primer in the PCR amplification of the KTI1 terminator(PSO400362) from the soybean genome. An EcoRI recognition site (GAATTC)was added for subsequent cloning.

SEQ ID NO:44 is the oligonucleotide primer PSO332986Sac used as a senseprimer in the PCR amplification of the PIP1 terminator (PSO332986) fromthe soybean genome. A SacI recognition site (GAGCTC) was added forsubsequent cloning.

SEQ ID NO:45 is the oligonucleotide primer PSO332986Eco used as anantisense primer in the PCR amplification of the PIP1 terminator(PSO332986) from the soybean genome. An EcoRI recognition site (GAATTC)was added for subsequent cloning.

SEQ ID NO:46 is the oligonucleotide primer PSO333268Sac used as a senseprimer in the PCR amplification of the EF1A2 terminator (PSO333268) fromthe soybean genome. A SacI recognition site (GAGCTC) was added forsubsequent cloning.

SEQ ID NO:47 is the oligonucleotide primer PSO333268Eco used as anantisense primer in the PCR amplification of the EF1A2 terminator(PSO333268) from the soybean genome. An EcoRI recognition site (GAATTC)was added for subsequent cloning.

SEQ ID NO:48 is the oligonucleotide primer PSO333209Sac used as a senseprimer in the PCR amplification of the MTH1 terminator (PSO333209) fromthe soybean genome. A SacI recognition site (GAGCTC) was added forsubsequent cloning.

SEQ ID NO:49 is the oligonucleotide primer PSO333209Eco used as anantisense primer in the PCR amplification of the MTH1 terminator(PSO333209) from the soybean genome. An EcoRI recognition site (GAATTC)was added for subsequent cloning.

SEQ ID NO:50 is the antisense oligo dT primer 3UTR-1 used to synthesizefirst strand cDNA from polyadenylated mRNA. A non-specific tail sequenceincluded on the 5′ end of the primer will be used as a priming site forsubsequent PCR.

SEQ ID NO:51 is the antisense primer 3UTR-2, which is specific to thetail sequence in primer 3UTR-1.

SEQ ID NO:52 is the antisense primer 3UTR-3, which is specific to aregion downstream of terminators MYB2, KTI1, PIP1, EF1A2, and MTH1 intheir respective constructs QC339, QC340, QC350, QC351, and QC352.

SEQ ID NO:53 is the sense primer SAMS-L, which is specific to anS-adenosylmethionine synthetase (SAMS) gene and is used in a diagnosticPCR to check for soybean genomic DNA presence in total RNA or cDNA.

SEQ ID NO:54 is the antisense primer SAMS-L2, which is specific to anS-adenosylmethionine synthetase (SAMS) gene and is used in a diagnosticPCR to check for soybean genomic DNA presence in total RNA or cDNA.

SEQ ID NO:55 is the antisense primer SAMS-A1, which is specific to theSAMS promoter used in constructs QC339, QC340, QC350, QC351, and QC352.

SEQ ID NO:56 is the antisense primer SAMS-A2, which is specific to theSAMS promoter.

SEQ ID NO:57 is the sense primer YFP-1, which is specific to theZS-YELLOW1 N1 (YFP) gene used in constructs QC339, QC340, QC350, QC351,and QC352.

SEQ ID NO:58 is the antisense primer YFP-2, which is specific to the YFPgene.

SEQ ID NO:59 is the sense primer YFP-3, which is specific to the YFPgene.

SEQ ID NO:60 is the antisense primer YFP-A, which is specific to the YFPgene.

SEQ ID NO:61 is the sense primer UBQ-S2, which is specific to thesoybean UBQ promoter used in constructs QC339, QC340, QC350, QC351, andQC352.

SEQ ID NO:62 is the sense primer SAMS-48F used in quantitative PCRanalysis of SAMS:ALS transgene copy numbers.

SEQ ID NO:63 is the FAM labeled (fluorescein) DNA probe SAMS-88T used inquantitative PCR analysis of SAMS:ALS transgene copy numbers.

SEQ ID NO:64 is the antisense primer SAMS-134R used in quantitative PCRanalysis of SAMS:ALS transgene copy numbers.

SEQ ID NO:65 is the sense primer YFP-67F used in quantitative PCRanalysis of YFP transgene copy numbers.

SEQ ID NO:66 is the FAM labeled (fluorescein) DNA probe YFP-88T used inquantitative PCR analysis of YFP transgene copy numbers.

SEQ ID NO:67 is the antisense primer YFP-130R used in quantitative PCRanalysis of YFP transgene copy numbers.

SEQ ID NO:68 is the sense primer HSP-F1 used as an endogenous controlgene primer HSP-F1 in quantitative PCR analysis of transgene copynumbers.

SEQ ID NO:69 is the VIC-labeled DNA probe HSP used as an endogenouscontrol gene probe in quantitative PCR analysis of transgene copynumbers.

SEQ ID NO:70 is the antisense primer HSP-R1 used as an endogenouscontrol gene primer in quantitative PCR analysis of transgene copynumbers.

SEQ ID NO:71 is the sense primer SamsPro-F used in quantitative RT-PCRanalysis of SAMS promoter transcripts.

SEQ ID NO:72 is the FAM-labeled (fluorescein) DNA MGB probe SamsPro-Tused in quantitative RT-PCR analysis of SAMS promoter transcripts.

SEQ ID NO:73 is the antisense primer SamsPro-R used in quantitativeRT-PCR analysis of SAMS promoter transcripts.

SEQ ID NO:74 is the sense primer YFP-139F used in quantitative RT-PCRanalysis of YFP transgene transcripts.

SEQ ID NO:75 is the FAM-labeled (fluorescein) DNA MGB probe YFP-160Tused in quantitative RT-PCR analysis of YFP transgene transcripts.

SEQ ID NO:76 is the antisense primer YFP-195R used in quantitativeRT-PCR analysis of YFP transgene transcripts.

SEQ ID NO:77 is the sense primer PSO323364S1 used for RT-PCR analysis ofendogenous gene PSO323364 transcripts.

SEQ ID NO:78 is the antisense primer PSO323364R1 used for RT-PCRanalysis of endogenous gene PSO323364 transcripts.

SEQ ID NO:79 is the sense primer PSO400362S1 used for RT-PCR analysis ofendogenous gene PSO400362 transcripts.

SEQ ID NO:80 is the antisense primer P50400362R1 used for RT-PCRanalysis of endogenous gene PSO400362 transcripts.

SEQ ID NO:81 is the sense primer PSO332982F used for RT-PCR analysis ofendogenous gene PSO332986 transcripts.

SEQ ID NO:82 is the antisense primer PSO332986JK-A used for RT-PCRanalysis of endogenous gene PSO332986 transcripts.

SEQ ID NO:83 is the sense primer PSO333268F used for RT-PCR analysis ofendogenous gene PSO333268 transcripts.

SEQ ID NO:84 is the antisense primer PSO333268R used for RT-PCR analysisof endogenous gene PSO333268 transcripts.

SEQ ID NO:85 is the sense primer PSO333209F used for RT-PCR analysis ofendogenous gene PSO333209 transcripts.

SEQ ID NO:86 is the antisense primer PSO333209JK-A used for RT-PCRanalysis of endogenous gene PSO333209 transcripts.

SEQ ID NO:87 is the recombination site attL1 sequence in the Gatewaycloning system (Invitrogen).

SEQ ID NO:88 is the recombination site attL2 sequence in the Gatewaycloning system (Invitrogen).

SEQ ID NO:89 is the recombination site attR1 sequence in the Gatewaycloning system (Invitrogen).

SEQ ID NO:90 is the recombination site attR2 sequence in the Gatewaycloning system (Invitrogen).

SEQ ID NO:91 is the recombination site attB1 sequence in the Gatewaycloning system (Invitrogen).

SEQ ID NO:92 is the recombination site attB2 sequence in the Gatewaycloning system (Invitrogen).

SEQ ID NO:93 is the VIC-labeled (fluorescein) DNA MGB probe ATPS-117Tused as the endogenous control in quantitative RT-PCR analysis of YFPand SAMS promoter transcripts.

SEQ ID NO:94 is the sequence of primer PSO323364UTR2.

SEQ ID NO:95 is the sequence of primer PSO323364UTR3.

SEQ ID NO:96 is the sequence of primer PSO323364UTR4.

SEQ ID NO:97 is the sequence of primer PSO323364UTR5.

SEQ ID NO:98 is the sequence of primer PSO323364UTR6.

SEQ ID NO:99 is the sequence of primer PSO323364UTR7.

SEQ ID NO:100 is the sequence of primer PSO400362UTR2.

SEQ ID NO:101 is the sequence of primer PSO400362UTR3.

SEQ ID NO:102 is the sequence of primer PSO400362UTR4.

SEQ ID NO:103 is the sequence of primer PSO400362UTR5.

SEQ ID NO:104 is the sequence of primer PSO400362UTR6.

SEQ ID NO:105 is the sequence of primer PSO400362UTR7.

SEQ ID NO:106 is the sequence of primer PSO332986UTR2.

SEQ ID NO:107 is the sequence of primer PSO332986UTR3.

SEQ ID NO:108 is the sequence of primer PSO332986UTR4.

SEQ ID NO:109 is the sequence of primer PSO332986UTR5.

SEQ ID NO:110 is the sequence of primer PSO332986UTR6.

SEQ ID NO:111 is the sequence of primer PSO332986UTR7.

SEQ ID NO:112 is the sequence of primer PSO333268UTR2.

SEQ ID NO:113 is the sequence of primer PSO333268UTR3.

SEQ ID NO:114 is the sequence of primer PSO333268UTR4.

SEQ ID NO:115 is the sequence of primer PSO333268UTR5.

SEQ ID NO:116 is the sequence of primer PSO333268UTR6.

SEQ ID NO:117 is the sequence of primer PSO333268UTR7.

SEQ ID NO:118 is the sequence of primer PSO333209UTR2.

SEQ ID NO:119 is the sequence of primer PSO333209UTR3.

SEQ ID NO:120 is the sequence of primer PSO333209UTR4.

SEQ ID NO:121 is the sequence of primer PSO333209UTR5.

SEQ ID NO:122 is the sequence of primer PSO333209UTR6.

SEQ ID NO:123 is the sequence of primer PSO333209UTR7.

SEQ ID NO:124 is the nucleotide sequence of the PSO323364 MYB2Lterminator.

SEQ ID NO:125 is the nucleotide sequence of the PSO400362 KTI1 Lterminator.

SEQ ID NO:126 is the nucleotide sequence of the PSO332986 PIP1 Lterminator.

SEQ ID NO:127 is the nucleotide sequence of the PSO333268 EF1A2Lterminator.

SEQ ID NO:128 is the nucleotide sequence of the PSO333209 MTH1Lterminator.

FIG. 1 shows the logarithm of relative gene expression quantificationsof five soybean genes PSO323364 (MYB2), PSO400362 (KTI1), PSO332986(PIP1), PSO333268 (EF1A2), and PSO333209 (MTH1) in 14 different soybeantissues by quantitative RT-PCR. The gene expression profiles indicatethat MYB2 is predominately expressed in flowers; KTI1 is predominatelyexpressed in developing seeds; and PIP1, EF1A2, and MTH1 are expressedsimilarly in all evaluated tissues.

FIG. 2 shows the maps of plasmids A) QC315, B) QC327, C) QC324i, and D)QC339. QC327 was made from QC315 bp replacing the NOS terminator inQC315 with the MYB2 terminator. The UBQ:YFP:MYB2 cassette in QC327 waslinked to the SAMS:HRA cassette in QC324i to make the final constructQC339.

FIG. 3 shows the maps of the following transformation ready constructs:A) QC340 for the KTI1 terminator, B) QC350 for the PIP1 terminator, C)QC351 for the EF1A2 terminator, and D) QC352 for the MTH1 terminator.

FIG. 4 shows the expression of YFP (yellow fluorescent protein) inrepresentative flower, leaf, stem, root, and pod/seed tissues oftransgenic plants derived from the terminator constructs QC339 (MYB2),QC340 (KTI1), QC350 (PIP1), QC351 (EF1A2), and QC352 (MTH1).

FIG. 5 shows the map of predicted terminator transgenes in plants andthe positions of primers used in RT-PCR and PCR analyses of transgeneexpression and RNA transcription termination.

FIG. 6 shows YFP reporter gene expression in four representativetransgenic events (samples 1, 2, 3, and 4) for each of the fiveterminators, MYB2, KTI1, PIP1, EF1A2, and MTH1. A) An RT-PCR check ofgenomic DNA contamination in RNA samples using primers SAMS-L/SAMS-L2.No genomic DNA-specific band was amplified from any of the RNA samples,and all RNA samples produced the RNA-specific band. B) A YFP expressioncheck by RT-PCR with YFP1/YFP-2 primers. YFP transcripts were detectedin all transgenic RNA samples. The two negative controls, wild type RNA(wt) and no template control (−), and the positive controls QC393 (+)and QC350 (+) all worked as expected.

FIG. 7 shows the transcription termination check by RT-PCR of the fivenovel terminators, MYB2, KTI1, PIP1, EF1A2, and MTH1, and a controlterminator PIN2. Wild type RNA (wt) and no template (−) were used asnegative controls. QC393, QC340, QC350, QC351, and QC352 were used aspositive controls (+). The same YFP-3 primer was used as the senseprimer. Three primers, 3UTR-3, SAMS-A1, SAMS-A2, progressivelydownstream of the terminator, were used as antisense primers for threeRT-PCR assays (A, B, and C, respectively). The sizes of the expectedRT-PCR bands are provided. A non-specific band was amplified from wildtype RNA in the first RT-PCR (A).

FIGS. 8A and 8B show the transcription termination check by RT-PCR offive endogenous genes from which the MYB2, KTI1, PIP1, EF1A2, and MTH1terminators were cloned. Gene-specific primers used in the analysis aredescribed in EXAMPLE 7. A) Two RT-PCR assays were performed on thefollowing three templates: wild type plantlet RNA as the target, H₂O asthe negative control, and genomic DNA as the positive control, with twosets of gene-specific primers for each terminator gene. RT-PCR-1 wasspecific to mRNA, while RT-PCR-2 was specific to RNA transcriptionread-through. Specific bands were detected with both RT-PCR assays forthe three constitutive genes MTH1, EF1A2, and PIP1, but not for theflower-specific gene MYB2 or the seed-specific gene KTI1. B) Wild typeflower and seed RNA were used in similar RT-PCR assays as the targettemplates to check the flower-specific gene MYB2 and the seed-specificgene KTI1. Specific bands were detected with both RT-PCR assays fromflower RNA for the flower-specific gene MYB2 and from seed RNA for theseed-specific gene KTI1. The RT-PCR with the SAMS-L/SAMS-L2 primer setwas done to check the flower RNA and seed RNA for genomic DNAcontamination.

FIG. 9 shows the RT-PCR amplification of 3′ UTR from transgenic plantshaving the MYB2, KTI1, PIP1, EF1A2, and MTH1 terminators. The firststrand cDNA was first made with an oligo dT primer 3UTR-1 by reversetranscription. Then the YFP-3/3UTR-2 primer set was used to amplify thepoly (A) containing 3UTR by PCR for subsequent cloning and sequencing.No specific band was amplified from the wild type (wt) and no template(−) negative controls.

FIG. 10 shows the relative positions of the single forward primerPSO323364S1 (SEQ ID NO:77) and the seven reverse primers PSO323364Eco(SEQ ID NO:41), PSO323364UTR2 (SEQ ID NO:94), PSO323364UTR3 (SEQ IDNO:95), PSO323364UTR4 (SEQ ID NO:96), PSO323364UTR5 (SEQ ID NO:97),PSO323364UTR6 (SEQ ID NO:98), and PSO323364UTR7 (SEQ ID NO:99) specificto the RNA transcript and the genomic DNA of gene PSO323364. Theseprimers were designed to check if the observed transcription readthrough of endogenous genes would stop and at what point. Primers weredesigned similarly for each of the other four genes.

FIGS. 11A and 11B show the results of seven RT-PCR experiments for eachof the five genes, PSO323364 (MYB2), PSO400362 (KTI1), PSO332986 (PIP1),PSO333268 (EF1A2), and PSO333209 (MTH1).

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of all patents, patent applications, and publicationscited herein are incorporated by reference in their entirety.

Definitions

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

The term “invention” or “present invention” as used herein is not meantto be limiting to any one specific embodiment of the invention butapplies generally to any and all embodiments of the invention asdescribed in the claims and specification.

In the context of this disclosure, a number of terms and abbreviationsare used. The following definitions are provided.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Polymerase chain reaction” is abbreviated PCR.

“Quantitative reverse transcription polymerase chain reaction” isabbreviated qRT-PCR.

“Reverse transcription polymerase chain reaction” is abbreviated RT-PCR.

As used herein, “GM-MYB2 terminator” or “MYB2 terminator” refer to the3′ untranslated sequence downstream of the coding region of the Glycinemax PSO323364 gene, which encodes a putative polypeptide withsignificant homology to MYB transcription factors (Uimari and Strommer,Plant J. 12 (6), 1273-1284 (1997)). “GM-KTI1 terminator” or “KTI1terminator” refer to the 3′ untranslated sequence downstream of thecoding region of the Glycine max PSO400362 gene, which encodes aputative polypeptide with significant homology to Kunitz trypsininhibitors (Jofuko and Goldberg, Plant Cell 1 (11), 1079-1093 (1989)). A“GM-PIP1 terminator” or “PIP1 terminator” refer to the 3′ untranslatedsequence downstream of the coding region of the Glycine max PSO332986gene, which encodes a putative polypeptide with significant homology toplasma membrane intrinsic proteins (Uehlein et al., Phytochemistry 68(1), 122-129 (2007)). A “GM-EF1A2 terminator” or “EF1A2 terminator”refer to the 3′ untranslated sequence downstream of the coding region ofthe Glycine max PSO333268 gene, which encodes a putative polypeptidewith significant homology to translation elongation factor EF-1α genesidentified in various species, including soybean (Aguilar et al., PlantMol. Biol. 17 (3), 351-360 (1991)). A “GM-MTH1 terminator” or “MTH1terminator” refer to the 3′ untranslated sequence downstream of thecoding region of the Glycine max PSO333209 gene, which encodes aputative polypeptide with significant homology to metallothionein-likeproteins (Munoz et al., Physiol. Plantarum 104, 273-279 (1998)).

The terminator nucleotide sequences are useful in combinations withdifferent promoters in regulating the expression of any heterologousnucleotide sequence in a host plant in order to alter the phenotype of aplant.

Various changes in phenotype are of interest including, but not limitedto, modifying the fatty acid composition in a plant, altering the aminoacid content of a plant, altering a plant's pathogen defense mechanism,and the like. These results can be achieved by providing expression ofheterologous products or increased expression of endogenous products inplants. Alternatively, the results can be achieved by providing for areduction of expression of one or more endogenous products, particularlyenzymes or cofactors in the plant. These changes result in a change inphenotype of the transformed plant.

A “marketable trait”, also referred to herein as a “marketable planttrait” or “commercial trait” or “commercially desirable trait”, is anytrait of importance to the commercial markets and interests of thoseinvolved in the development of the crop, wherein the marketable trait isevaluated in a fertile, mature plant. A marketable or commercial traitcan include, without limitation, disease resistance, herbicideresistance, insect resistance, carbohydrate metabolism, fatty acidmetabolism, amino acid metabolism, plant development, plant growthregulation, yield improvement, drought resistance, cold resistance, heatresistance, and salt resistance.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge. In addition, as our understanding ofagronomic characteristics and traits, such as yield and heterosis,increases, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, but arenot limited to, those genes involved in information, such as zincfingers; those involved in communication, such as kinases; and thoseinvolved in housekeeping, such as heat shock proteins. More specificcategories of transgenes, for example, include, but are not limited to,genes involved in important traits for agronomics, insect resistance,disease resistance, herbicide resistance, sterility, grain or seedcharacteristics, and commercial products. Genes of interest include,generally, those involved in oil, starch, carbohydrate, or nutrientmetabolism as well as those affecting seed size, plant development,plant growth regulation, and yield improvement. Plant development andgrowth regulation also refer to the development and growth regulation ofvarious parts of a plant, such as the flower, seed, root, leaf andshoot.

Other commercially desirable traits involve genes and proteinsconferring cold, heat, salt, or drought resistance.

Disease and/or insect resistance genes may confer resistance to peststhat significantly decrease yield, such as for example, anthracnose;soybean mosaic virus; soybean cyst nematode; root-knot nematode; thefungal agents that cause brown leaf spot, Downy mildew, purple seedstain, seed decay, and seedling diseases; and the bacterium Pseudomonassyringae pv. Glycinea that causes bacterial blight. Genes involved ininsect resistance include, for example, Bacillus thuringiensis toxicprotein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,723,756;5,593,881; and Geiser et al., Gene 48:109 (1986)); lectins (Van Damme etal., Plant Mol. Biol. 24:825 (1994)); and the like.

Herbicide resistance traits may include genes conferring resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides. The S4 and/or HRAmutations in the acetolactate synthase ALS gene, for example, conferresistance to the herbicide chlorsulfuron. Glyphosate acetyl transferase(GAT) is an N-acetyltransferase from Bacillus licheniformis that wasoptimized by gene shuffling for acetylation of the broad spectrumherbicide, glyphosate, forming the basis of a novel mechanism ofglyphosate tolerance in transgenic plants (Castle et al., Science 304,1151-1154 (2004)).

Antibiotic resistance genes include, for example, neomycinphosphotransferase (npt) and hygromycin phosphotransferase (hpt). Twoneomycin phosphotransferase genes are used in selection of transformedorganisms: the neomycin phosphotransferase I (nptI) gene and theneomycin phosphotransferase II (nptII) gene, the latter of which is morewidely used. The nptII gene was initially isolated from the transposonTn5 present in the bacterium strain Escherichia coli K12 (Beck et al.,Gene 19, 327-36 (1982)). The gene codes for the aminoglycoside3′-phosphotransferase (denoted aph(3′)-II or NPTII) enzyme, whichinactivates by phosphorylation a range of aminoglycoside antibioticssuch as kanamycin, neomycin, geneticin, and paroromycin. NPTII is widelyused as a selectable marker for plant transformation. It is also used ingene expression and regulation studies in different organisms in partbecause N-terminal fusions can be constructed that retain enzymeactivity. NPTII protein activity can be detected by enzymatic assay. Inother detection methods, the modified substrates, the phosphorylatedantibiotics, are detected by thin-layer chromatography, dot-blotanalysis, or polyacrylamide gel electrophoresis. Plants such as maize,cotton, tobacco, Arabidopsis, flax, soybean, and many others have beensuccessfully transformed with the nptII gene.

The hygromycin phosphotransferase (denoted hpt, hph, or aphIV) gene wasoriginally derived from Escherichia coli (Gritz et al., Gene 25, 179-188(1983)). The gene codes for hygromycin phosphotransferase (HPT), whichdetoxifies the aminocyclitol antibiotic hygromycin B. A large number ofplants have been transformed with the hpt gene, and hygromycin B hasproved very effective in the selection of a wide range of plants,including monocots. Most plants, e.g. cereals, exhibit highersensitivity to hygromycin B than to kanamycin. Likewise, the hpt gene isused widely in selection of transformed mammalian cells. The sequence ofthe hpt gene has been modified for use in plant transformation.Deletions and substitutions of amino acid residues close to the carboxy(C)-terminus of the enzyme have increased the level of resistance incertain plants, such as tobacco. At the same time, the hydrophilicC-terminus of the enzyme has been maintained and may be essential forthe strong activity of HPT. HPT activity can be checked using anenzymatic assay. A non-destructive callus induction test can be used toverify hygromycin resistance.

Genes involved in plant growth and development have been identified inplants. One such gene, which is involved in cytokinin biosynthesis, isisopentenyl transferase (IPT). Cytokinin plays a critical role in plantgrowth and development by stimulating cell division and celldifferentiation (Sun et al., Plant Physiol. 131: 167-176 (2003)).

Calcium-dependent protein kinases (CDPK), a family of serine-threoninekinases found primarily in the plant kingdom, are likely to function assensor molecules in calcium-mediated signaling pathways. Calcium ionsare important secondary messengers during plant growth and development(Harper et al., Science 252, 951-954 (1993); Roberts et al., Curr OpinCell Biol 5, 242-246 (1993); Roberts et al., Annu Rev Plant Mol Biol 43,375-414 (1992)).

Nematode responsive protein (NRP) is produced by soybean upon theinfection of soybean cyst nematode. NRP has homology to ataste-modifying glycoprotein miraculin and the NF34 protein involved intumor formation and hyper response induction. NRP is believed tofunction as a defense-inducer in response to nematode infection(Tenhaken et al., BMC Bioinformatics 6:169 (2005)).

The quality of seeds and grains is reflected in traits such as levelsand types of fatty acids or oils (saturated and unsaturated), qualityand quantity of essential amino acids, and levels of carbohydrates.Therefore, commercial traits involving a gene or genes that increase theamino acids methionine and cysteine, two sulfur containing amino acidspresent in low amounts in soybeans, are of interest. Cystathionine gammasynthase (CGS) and serine acetyl transferase (SAT) are enzymes involvedin the synthesis of methionine and cysteine, respectively.

Other commercial traits can involve genes that increase, for example,monounsaturated fatty acids, such as oleic acid, in oil seeds. Soybeanoil contains high levels of polyunsaturated fatty acids and is moreprone to oxidation than oils with higher levels of monounsaturated andsaturated fatty acids. High oleic soybean seeds can be prepared byrecombinant manipulation of the activity of oleoyl 12-desaturase (Fad2),and high oleic soybean oil can then be used in applications that requirea high degree of oxidative stability, such as cooking for a long periodof time at an elevated temperature.

Raffinose saccharides accumulate in significant quantities in the edibleportion of many economically significant crop species, such as soybean(Glycine max L. Merrill), sugar beet (Beta vulgaris), cotton (Gossypiumhirsutum L.), canola (Brassica sp.), and all of the major edibleleguminous crops including beans (Phaseolus sp.), chick pea (Cicerarietinum), cowpea (Vigna unguiculata), mung bean (Vigna radiata), peas(Pisum sativum), lentil (Lens culinaris) and lupine (Lupinus sp.).Although abundant in many species, raffinose saccharides are an obstacleto the efficient utilization of some economically important cropspecies. Thus, downregulation of the expression of the enzymes involvedin raffinose saccharide synthesis, such as galactinol synthase, forexample, would be a desirable trait.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may include, butare not limited to, promoters, translation leader sequences, introns,and polyadenylation recognition sequences.

The term “expression”, as used herein, refers to the production of afunctional end-product e.g., an mRNA or a protein (precursor or mature).“Altering expression” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ significantlyfrom the amount of the gene product(s) produced by the correspondingwild-type organisms (i.e., expression is increased or decreased).

Expression or overexpression of a gene involves transcription of thegene and translation of the mRNA into a precursor or mature protein.“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Co-suppression” refers to the production ofsense RNA transcripts capable of suppressing the expression ortranscript accumulation of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020). The mechanism ofco-suppression may be at the DNA level (such as DNA methylation), at thetranscriptional level, or at post-transcriptional level.

Co-suppression constructs in plants previously have been designed byfocusing on overexpression of a nucleic acid sequence having homology toan endogenous mRNA, in the sense orientation, which results in thereduction of all RNA having homology to the overexpressed sequence (seeVaucheret et al., Plant J. 16:651-659 (1998); and Gura, Nature404:804-808 (2000)). The overall efficiency of this phenomenon is low,and the extent of the RNA reduction is widely variable. Recent work hasdescribed the use of “hairpin” structures that incorporate all, or part,of an mRNA encoding sequence in a complementary orientation that resultsin a potential “stem-loop” structure for the expressed RNA (PCTPublication No. WO 99/53050 published on Oct. 21, 1999; and PCTPublication No. WO 02/00904 published on Jan. 3, 2002). This increasesthe frequency of co-suppression in the recovered transgenic plants.Another variation describes the use of plant viral sequences to directthe suppression, or “silencing”, of proximal mRNA encoding sequences(PCT Publication No. WO 98/36083 published on Aug. 20, 1998). Currentdata has suggested that dsRNA mediated mRNA cleavage may have been theconserved mechanism underlying these gene silencing phenomena (Elmayanet al., Plant Cell 10:1747-1757 (1998); Galun, In Vitro Cell. Dev. Biol.Plant 41(2):113-123 (2005); Pickford et al., Cell. Mol. Life Sci.60(5):871-882 (2003)).

The terms “fragment (or variant) that is functionally equivalent” and“functionally equivalent fragment (or variant)” are used interchangeablyherein. These terms refer to a portion or subsequence or variant of theterminator sequence of the present invention in which the ability toterminate transcription is retained. Fragments and variants can beobtained via methods such as site-directed mutagenesis and syntheticconstruction. Recombinant DNA constructs can be designed for use inco-suppression or antisense by linking a promoter, a heterologousnucleotide sequence, and a terminator fragment or variant thereof.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene”, “recombinant DNA construct”, or“recombinant expression construct”, which are used interchangeably,refer to any gene that is not a native gene, comprising regulatory andcoding sequences that are not found together in nature. Accordingly, achimeric gene may comprise regulatory sequences and coding sequencesthat are derived from different sources, or regulatory sequences andcoding sequences derived from the same source, but arranged in a mannerdifferent than that found in nature. “Endogenous gene” refers to anative gene in its natural location in the genome of an organism. A“foreign” gene refers to a gene not normally found in the host organism,but that is introduced into the host organism by gene transfer. Foreigngenes can comprise native genes inserted into a non-native organism, orchimeric genes. A “transgene” is a gene that has been introduced intothe genome by a transformation procedure.

A “heterologous nucleic acid fragment” or “heterologous nucleotidesequence” refers to a nucleotide sequence that is not naturallyoccurring with the plant terminator sequence of the invention. Whilethis nucleotide sequence is heterologous to the terminator sequence, itmay be homologous, or native, or heterologous, or foreign, to the planthost.

An “intron” is an intervening sequence in a gene that is transcribedinto RNA and then excised in the process of generating the mature mRNA.The term is also used for the excised RNA sequences. An “exon” is aportion of the sequence of a gene that is transcribed and is found inthe mature messenger RNA derived from the gene. An exon is notnecessarily a part of the sequence that encodes the final gene product.

An “isolated nucleic acid fragment” or “isolated polynucleotide” refersto a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA) thatis single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. An isolated polynucleotide inthe form of DNA may be comprised of one or more segments of cDNA,genomic DNA, or synthetic DNA.

The term “operably linked” refers to the association of nucleic acidsequences on a single polynucleotide so that the function of one isaffected by the other. For example, a promoter is operably linked with aheterologous nucleotide sequence, e.g. a coding sequence, when it iscapable of affecting the expression of that heterologous nucleotidesequence (i.e., the coding sequence is under the transcriptional controlof the promoter). A coding sequence can be operably linked to regulatorysequences in sense or antisense orientation.

A “plasmid” or “vector” is an extra chromosomal element often carryinggenes that are not part of the central metabolism of the cell, andusually in the form of circular double-stranded DNA fragments. Suchelements may be autonomously replicating sequences, genome integratingsequences, phage or nucleotide sequences, linear or circular, of asingle- or double-stranded DNA or RNA, derived from any source, in whicha number of nucleotide sequences have been joined or recombined into aunique construction which is capable of introducing an expressioncassette(s) into a cell. “Expression cassette” refers to a fragment ofDNA containing a foreign gene and having elements in addition to theforeign gene that allow for enhanced expression of that gene in aforeign host. “Transformation cassette” refers to a fragment of DNAcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.

“PCR” or “Polymerase Chain Reaction” is a technique for the synthesis oflarge quantities of specific DNA segments, consisting of a series ofrepetitive cycles (Perkin Elmer Cetus Instruments, Norwalk, Conn.).Typically, the double stranded DNA is heat denatured, and the twoprimers complementary to the 3′ boundaries of the target segment areannealed at low temperature and then extended at an intermediatetemperature. One set of these three consecutive steps comprises a cycle.

The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acidsequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment”are used interchangeably herein. These terms encompass nucleotidesequences and the like. A polynucleotide may be a polymer of RNA or DNAthat is single- or double-stranded, that optionally contains synthetic,non-natural or altered nucleotide bases. A polynucleotide in the form ofa polymer of DNA may be comprised of one or more segments of cDNA,genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by a single letterdesignation as follows: “A” for adenylate or deoxyadenylate (for RNA orDNA, respectively), “C” for cytidylate or deoxycytidylate, “G” forguanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. Functional RNAincludes, but is not limited to, transfer RNA (tRNA) and ribosomal RNA(rRNA). The promoter sequence consists of proximal and more distalupstream elements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence which can stimulatepromoter activity and may be an innate element of the promoter or aheterologous element inserted to enhance the level or tissue-specificityof a promoter. Promoters may be derived in their entirety from a nativegene, or be composed of different elements derived from differentpromoters found in nature, or even comprise synthetic DNA segments. Itis understood by those skilled in the art that different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. Promoters which cause a gene to be expressedin most cell types at most times are commonly referred to as“constitutive promoters”. It is further recognized that since in mostcases the exact boundaries of regulatory sequences have not beencompletely defined, DNA fragments of some variation may have identicalpromoter activity.

The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

The terms “recombinant construct”, “expression construct”, “chimericconstruct”, “construct”, and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialcombination of nucleic acid fragments, e.g., regulatory and codingsequences that are not found together in nature. For example, arecombinant construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. Such aconstruct may be used by itself or may be used in conjunction with avector. If a vector is used, then the choice of vector is dependent uponthe method that will be used to transform host cells as is well known tothose skilled in the art. For example, a plasmid vector can be used. Theskilled artisan is well aware of the genetic elements that must bepresent on the vector in order to successfully transform, select andpropagate host cells comprising any of the isolated nucleic acidfragments of the invention. The skilled artisan will also recognize thatdifferent independent transformation events will result in differentlevels and patterns of expression (Jones et al., EMBO J. 4:2411-2418(1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.et al., In Molecular Cloning: A Laboratory Manual; 2^(nd) ed.; ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989(hereinafter “Sambrook et al., 1989”) or Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl,K., Eds.; In Current Protocols in Molecular Biology; John Wiley andSons: New York, 1990 (hereinafter “Ausubel et al., 1990”).

“RNA transcript” refers to a product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When an RNAtranscript is a perfect complementary copy of a DNA sequence, it isreferred to as a primary transcript, or it may be an RNA sequencederived from posttranscriptional processing of a primary transcript andis referred to as a mature RNA. “Messenger RNA” (“mRNA”) refers to RNAthat is without introns and that can be translated into protein by thecell. “cDNA” refers to a DNA that is complementary to and synthesizedfrom an mRNA template using the enzyme reverse transcriptase. The cDNAcan be single-stranded or converted into the double-stranded by usingthe Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNAtranscript that includes mRNA and so can be translated into proteinwithin a cell or in vitro. “Antisense RNA” refers to a RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks expression or transcript accumulation of a targetgene (U.S. Pat. No. 5,107,065). The complementarity of an antisense RNAmay be with any part of the specific gene transcript, i.e. at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, orother RNA that may not be translated yet has an effect on cellularprocesses.

A “substantially homologous sequence” refers to variants of thedisclosed sequences such as those that result from site-directedmutagenesis, as well as synthetically derived sequences. A substantiallyhomologous sequence of the present invention also refers to thosefragments of a particular terminator nucleotide sequence disclosedherein that operate to terminate transcription of an operably linkedheterologous nucleic acid fragment. These terminator fragments willcomprise at least about 20 contiguous nucleotides, preferably at leastabout 50 contiguous nucleotides, more preferably at least about 75contiguous nucleotides, even more preferably at least about 100contiguous nucleotides of the particular terminator nucleotide sequencedisclosed herein. Such fragments may be obtained by use of restrictionenzymes to cleave the naturally occurring terminator nucleotidesequences disclosed herein; by synthesizing a nucleotide sequence fromthe naturally occurring terminator DNA sequence; or may be obtainedthrough the use of PCR technology. See particularly, Mullis et al.,Methods Enzymol. 155:335-350 (1987), and Higuchi, R. In PCR Technology:Principles and Applications for DNA Amplifications; Erlich, H. A., Ed.;Stockton Press Inc.: New York, 1989. Again, variants of these terminatorfragments, such as those resulting from site-directed mutagenesis, areencompassed by the compositions of the present invention.

The terms “substantially similar” and “corresponding substantially” asused herein refer to nucleic acid fragments, particularly terminatorsequences, wherein changes in one or more nucleotide bases do notsubstantially alter the ability of the terminator to terminatetranscription. These terms also refer to modifications, includingdeletions and variants, of the nucleic acid sequences of the instantinvention by way of deletion or insertion of one or more nucleotidesthat do not substantially alter the functional properties of theresulting terminator relative to the initial, unmodified terminator. Itis therefore understood, as those skilled in the art will appreciate,that the invention encompasses more than the specific exemplarysequences.

Moreover, the skilled artisan recognizes that substantially similarnucleic acid sequences encompassed by this invention are also defined bytheir ability to hybridize, under moderately stringent conditions (forexample, 0.5×SSC, 0.1% SDS, 60° C.) with the sequences exemplifiedherein, or to any portion of the nucleotide sequences reported hereinand which are functionally equivalent to the terminator of theinvention. Estimates of such homology are provided by either DNA-DNA orDNA-RNA hybridization under conditions of stringency as is wellunderstood by those skilled in the art (Hames and Higgins, Eds.; InNucleic Acid Hybridisation; IRL Press: Oxford, U.K., 1985). Stringencyconditions can be adjusted to screen for moderately similar fragments,such as homologous sequences from distantly related organisms, to highlysimilar fragments, such as genes that duplicate functional enzymes fromclosely related organisms. Post-hybridization washes partially determinestringency conditions. One set of conditions uses a series of washesstarting with 6×SSC, 0.5% SDS at room temperature for 15 min, thenrepeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeatedtwice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. Another set ofstringent conditions uses higher temperatures in which the washes areidentical to those above except for the temperature of the final two 30min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another set ofhighly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDSat 65° C.

Preferred substantially similar nucleic acid sequences encompassed bythis invention are those sequences that are 80% identical to the nucleicacid fragments reported herein or which are 80% identical to any portionof the nucleotide sequences reported herein. More preferred are nucleicacid fragments which are 90% identical to the nucleic acid sequencesreported herein, or which are 90% identical to any portion of thenucleotide sequences reported herein. Most preferred are nucleic acidfragments which are 95% identical to the nucleic acid sequences reportedherein, or which are 95% identical to any portion of the nucleotidesequences reported herein. It is well understood by one skilled in theart that many levels of sequence identity are useful in identifyingrelated polynucleotide sequences. Useful examples of percent identitiesare those listed above, or also preferred is any integer percentage from80% to 100%, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 and 99%.

Sequence alignments and percent similarity calculations may bedetermined using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.) or using the AlignXprogram of the Vector NTI bioinformatics computing suite (Invitrogen,Carlsbad, Calif.). Multiple alignment of the sequences are performedusing the Clustal method of alignment (Higgins and Sharp, CABIOS5:151-153 (1989)) with the default parameters (GAP PENALTY=10, GAPLENGTH PENALTY=10). Default parameters for pairwise alignments andcalculation of percent identity of protein sequences using the Clustalmethod are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Fornucleic acids these parameters are GAP PENALTY=10, GAP LENGTHPENALTY=10, KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. A“substantial portion” of an amino acid or nucleotide sequence comprisesenough of the amino acid sequence of a polypeptide or the nucleotidesequence of a gene to afford putative identification of that polypeptideor gene, either by manual evaluation of the sequence by one skilled inthe art, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Altschul, S. F. et al., J. Mol. Biol.215:403-410 (1993)) and Gapped Blast (Altschul, S. F. et al., NucleicAcids Res. 25:3389-3402 (1997)). The “BLASTN method of alignment” refersto a BLAST program that compares a nucleotide query sequence against anucleotide sequence database.

As stated herein, “suppression” refers to a reduction of the level ofenzyme activity or protein functionality (e.g., a phenotype associatedwith a protein) detectable in a transgenic plant when compared to thelevel of enzyme activity or protein functionality detectable in anon-transgenic or wild type plant with the native enzyme or protein. Thelevel of enzyme activity in a plant with the native enzyme is referredto herein as “wild type” activity. The level of protein functionality ina plant with the native protein is referred to herein as “wild type”functionality. The term “suppression” includes lower, reduce, decline,decrease, inhibit, eliminate, and prevent. This reduction may be due toa decrease in translation of the native mRNA into an active enzyme orfunctional protein. It may also be due to the transcription of thenative DNA into decreased amounts of mRNA and/or to rapid degradation ofthe native mRNA. The term “native enzyme” refers to an enzyme that isproduced naturally in a non-transgenic or wild type cell. The terms“non-transgenic” and “wild type” are used interchangeably herein.

“Transcription terminator”, “3′ non-coding sequences”, “terminationsequences”, or “terminator” refer to DNA sequences located downstream ofa coding sequence, including polyadenylation recognition sequences andother sequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al., Plant Cell1:671-680 (1989).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Thus, a “transgenicplant cell’ as used herein refers to a plant cell containing thetransformed nucleic acid fragments. The preferred method of soybean celltransformation is the use of particle-accelerated or “gene gun”transformation technology (Klein et al., Nature (London) 327:70-73(1987); U.S. Pat. No. 4,945,050).

“Transient expression” refers to the temporary expression of a gene,often a reporter gene such as β-glucuronidase (GUS) or any of thefluorescent protein genes, GFP, ZS-YELLOW1 N1, AM-CYAN1, and DS-RED, inselected certain cell types of the host organism in which the transgenicgene is introduced temporally by a transformation method. Thetransformed material of the host organism is subsequently discardedafter the transient gene expression assay.

The “translation leader sequence” refers to a DNA sequence locatedbetween the promoter sequence of a gene and the coding sequence. Thetranslation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to mRNA, mRNAstability, or translation efficiency. Examples of translation leadersequences have been described (Turner, R. and Foster, G. D., MolecularBiotechnology 3:225 (1995)).

This invention concerns isolated terminators of a MYB familytranscription factor (MYB2), a Kunitz trypsin inhibitor (KTI1), a plasmamembrane intrinsic protein (PIP1), a translation elongation factor(EF-1A), and a metallothionein protein (MTH1).

This invention concerns an isolated polynucleotide comprising aterminator wherein said terminator comprises the nucleotide sequence setforth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, or SEQID NO:128; a full-length complement of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ IDNO:126, SEQ ID NO:127, or SEQ ID NO:128; or a nucleotide sequence havingat least 90% sequence identity, based on the BLASTN method of alignment,when compared to the sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ IDNO:126, SEQ ID NO:127, or SEQ ID NO:128.

A nucleic acid fragment that is functionally equivalent to an instantterminator is any nucleic acid fragment that is capable of terminatingthe transcription of a coding sequence or functional RNA in a similarmanner as the terminator. Thus, the invention also includes a nucleotidesequence comprising a fragment of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,SEQ ID NO:127, or SEQ ID NO:128; a full-length complement of a fragmentof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, or SEQ IDNO:128; or a nucleotide sequence having at least 90% sequence identity,based on the BLASTN method of alignment, when compared to the fragmentof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, or SEQ IDNO:128, or a full-length complement thereof, wherein the nucleotidesequence functions as a terminator.

The terminator activity of each of the soybean genomic DNA fragments ofSEQ ID NO:1, 2, 3, 4, and 5 was individually assessed by linking thefragment to a yellow fluorescence reporter gene, ZS-YELLOW1 N1 (YFP)which is controlled by a soybean ubiquitin gene promoter GM-UBQ (Matz etal., Nat. Biotechnol. 17:969-973 (1999)), transforming theUBQ:YFP:terminator expression cassette into soybean, and analyzing YFPexpression in various cell types of the transgenic plants (see EXAMPLES7 and 8). YFP expression was detected in all parts of the transgenicplants, though stronger expression was detected in fast growing tissuessuch as developing embryos and pods. These results indicated that thenucleic acid fragment functioned as a transcription terminator to addpolyadenylation tails on the YFP gene transcripts and to terminate YFPgene transcription.

It is clear from the disclosure set forth herein that one of ordinaryskill in the art could perform the following procedure:

1) operably linking the nucleic acid fragment containing a terminatorsequence of the invention to a suitable reporter gene; there are avariety of reporter genes that are well known to those skilled in theart, including the bacterial GUS gene, the firefly luciferase gene, andthe cyan, green, red, and yellow fluorescent protein genes; any gene forwhich an easy and reliable assay is available can serve as the reportergene.

2) transforming a chimeric promoter:reporter:terminator gene expressioncassette into an appropriate plant for expression of the reporter. Thereare a variety of appropriate plants which can be used as a host fortransformation that are well known to those skilled in the art,including the dicots, Arabidopsis, tobacco, soybean, oilseed rape,peanut, sunflower, safflower, cotton, tomato, potato, and cocoa and themonocots, corn, wheat, rice, barley, and palm.

3) testing for expression of the promoter:reporter:terminator in variouscell types of transgenic plant tissues, e.g., leaves, roots, flowers,seeds, transformed with the chimeric promoter:reporter:terminator geneexpression cassette by assaying for expression of the reporter geneproduct.

In another aspect, this invention concerns a recombinant DNA constructcomprising a promoter, at least one heterologous nucleic acid fragment,and any terminator, or combination of terminator elements, of thepresent invention, wherein the promoter, at least one heterologousnucleic acid fragment, and terminator(s) are operably linked.Recombinant DNA constructs can be constructed by operably linking thenucleic acid fragment of the invention, the terminator sequence setforth in SEQ ID NO:1, 2, 3, 4, 5, 124, 125, 126, 127, or 128 or afragment that is substantially similar and functionally equivalent toany portion of the nucleotide sequence set forth in SEQ ID NO:1, 2, 3,4, 5, 124, 125, 126, 127, or 128, to a heterologous nucleic acidfragment. Any heterologous nucleic acid fragment can be used to practicethe invention. The selection will depend upon the desired application orphenotype to be achieved. The various nucleic acid sequences can bemanipulated so as to provide for the nucleic acid sequences in theproper orientation.

In another embodiment, this invention concerns host cells comprisingeither the recombinant DNA constructs of the invention as describedherein or isolated polynucleotides of the invention as described herein.Examples of host cells which can be used to practice the inventioninclude, but are not limited to, yeast, bacteria, and plants.

Plasmid vectors comprising the instant recombinant DNA construct can beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host cells. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select, and propagate host cellscontaining the chimeric gene.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published, amongothers, for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No. 5,159,135);soybean (U.S. Pat. No. 5,569,834, U.S. Pat. No. 5,416,011); Brassica(U.S. Pat. No. 5,463,174); peanut (Cheng et al., Plant Cell Rep.15:653-657 (1996), McKently et al., Plant Cell Rep. 14:699-703 (1995));papaya (Ling et al., Bio/technology 9:752-758 (1991)); and pea (Grant etal., Plant Cell Rep. 15:254-258 (1995)). For a review of other commonlyused methods of plant transformation see Newell, C. A., Mol. Biotechnol.16:53-65 (2000). One of these methods of transformation usesAgrobacterium rhizogenes (Tepfler, M. and Casse-Delbart, F., Microbiol.Sci. 4:24-28 (1987)). Transformation of soybeans using direct deliveryof DNA has been published using PEG fusion (PCT Publication No. WO92/17598), electroporation (Chowrira et al., Mol. Biotechnol. 3:17-23(1995); Christou et al., Proc. Natl. Acad. Sci. U.S.A. 84:3962-3966(1987)), microinjection, or particle bombardment (McCabe et al.,Bio/Technology 6:923 (1988); Christou et al., Plant Physiol. 87:671-674(1988)).

There are a variety of methods for the regeneration of plants from planttissues. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated. The regeneration, development and cultivation of plantsfrom single plant protoplast transformants or from various transformedexplants is well known in the art (Weissbach and Weissbach, Eds.; InMethods for Plant Molecular Biology; Academic Press, Inc.: San Diego,Calif., 1988). This regeneration and growth process typically includesthe steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentor through the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of thepresent invention containing a desired polypeptide is cultivated usingmethods well known to one skilled in the art.

In addition to the above discussed procedures, practitioners arefamiliar with the standard resource materials which describe specificconditions and procedures for the construction, manipulation andisolation of macromolecules (e.g., DNA molecules, plasmids, etc.),generation of recombinant DNA fragments and recombinant expressionconstructs and the screening and isolating of clones, (see for example,Sambrook, J. et al., In Molecular Cloning: A Laboratory Manual; 2^(nd)ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.,1989; Maliga et al., In Methods in Plant Molecular Biology; Cold SpringHarbor Press, 1995; Birren et al., In Genome Analysis: Detecting Genes,1; Cold Spring Harbor: N.Y., 1998; Birren et al., In Genome Analysis:Analyzing DNA, 2; Cold Spring Harbor: N.Y., 1998; Clark, Ed., In PlantMolecular Biology: A Laboratory Manual; Springer: N.Y., 1997).

The skilled artisan will also recognize that different independenttransformation events will result in different levels and patterns ofexpression of the chimeric genes (Jones et al., EMBO J. 4:2411-2418(1985); De Almeida et al., Mol. Gen. Genetics 218:78-86 (1989)). Thus,multiple events must be screened in order to obtain lines displaying thedesired expression level and pattern. Such screening may be accomplishedby Northern analysis of mRNA expression, Western analysis of proteinexpression, or phenotypic analysis. Also of interest are seeds obtainedfrom transformed plants displaying the desired gene expression profile.

Another general application of the terminators of the invention is toconstruct chimeric genes that can be used to reduce expression of atleast one heterologous nucleic acid fragment in a plant cell. Toaccomplish this, a chimeric gene designed for gene silencing of aheterologous nucleic acid fragment can be constructed by linking thefragment to a promoter of choice and a terminator of the presentinvention. (See U.S. Pat. No. 5,231,020, and PCT Publication No. WO99/53050, PCT Publication No. WO 02/00904, and PCT Publication No. WO98/36083, for methodology to block plant gene expression viacosuppression.) Alternatively, a chimeric gene designed to expressantisense RNA for a heterologous nucleic acid fragment can beconstructed by linking the fragment in reverse orientation to theterminator of the present invention. (See U.S. Pat. No. 5,107,065 formethodology to block plant gene expression via antisense RNA.) Eitherthe cosuppression or antisense chimeric gene can be introduced intoplants via transformation. Transformants wherein expression of theheterologous nucleic acid fragment is decreased or eliminated are thenselected.

This invention also concerns a method of expressing at least oneheterologous nucleic acid fragment in a plant cell which comprises:

-   -   (a) transforming a plant cell with the recombinant DNA construct        described herein;    -   (b) growing fertile mature plants from the transformed plant        cell of step (a);    -   (c) selecting plants containing a transformed plant cell wherein        the heterologous nucleic acid fragment is expressed.

Transformation and selection can be accomplished using methodswell-known to those skilled in the art including, but not limited to,the methods described herein.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

In the discussion below, parts and percentages are by weight, anddegrees are Celsius, unless otherwise stated. Sequences of promoters,cDNA, adaptors, terminators, and primers listed in this invention are inthe 5′ to 3′ orientation unless described otherwise. Techniques inmolecular biology were typically performed as described in Ausubel, F.M. et al. (In Current Protocols in Molecular Biology; John Wiley andSons: New York, (1990)) or Sambrook, J. et al. (In Molecular Cloning: ALaboratory Manual; 2^(nd) ed.; Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y., (1989)) (hereinafter “Sambrook et al., 1989”).

Example 1 Identification of Terminator Candidate Genes

Soybean expression sequence tags (EST) were generated by sequencingrandomly selected clones from cDNA libraries constructed from differentsoybean tissues. Multiple EST sequences could often be found withdifferent lengths representing the different regions of the same soybeangene. For those EST sequences representing the same gene that are foundmore frequently in one tissue-specific cDNA library than in another,there is a possibility that the represented gene could be atissue-preferred gene candidate. For example, EST sequences representingthe same gene that are found more frequently in a flower library than ina leaf library may indicate a flower-preferred gene candidate.Alternatively, if similar numbers of ESTs for the same gene are found invarious libraries constructed from different tissues, the representedgene could be a constitutively expressed gene. Multiple EST sequencesrepresenting the same soybean gene were compiled electronically, basedon their overlapping sequence homology, into a full length sequencerepresenting that unique gene. The assembled unique gene sequences werecollected, and the information was stored in searchable databases.

To identify strong constitutively expressed genes, database searcheswere performed to detect gene sequences found at similar frequenciesacross multiple tissue-specific libraries, such as leaf, root, flower,embryos, pod, etc. To identify tissue-specific genes, e.g. seed-specificgenes, searches were performed to look for gene sequences found at highfrequency in one tissue-specific library, such as a seed-specificlibrary, and at little to no frequency in other tissue-specificlibraries. Several constitutive and tissue-specific genes wereidentified as candidates for the cloning of novel promoters and/orterminators.

A more sensitive gene expression profiling methodology MPSS (MassParallel Signature Sequence) transcript profiling technique (Brenner etal., Proc. Natl. Acad. Sci. USA 97:1665-70 (2000)) was used to confirmthe gene expression profiles of the candidate genes. The MPSS technologyinvolves the generation of 17 base signature tags from mRNA samples thathave been reverse transcribed from poly A+ RNA isolated using standardmolecular biology techniques (Sambrook et al., 1989). The tags aresimultaneously sequenced and assigned to genes or ESTs. The abundance ofthese tags is given a number value that is normalized to parts permillion (PPM), which then allows the tag expression, or tag abundance,to be compared across different tissues. Thus, the MPSS platform can beused to determine the expression pattern of a particular gene and itsexpression levels in different tissues.

MPSS gene expression profiles were generated from different soybeantissues over time, and the profiles were accumulated in a searchabledatabase. Each candidate gene sequence was first used to search the MPSSdatabase to identify an MPSS tag that was identical to a 17 base pairregion in the 3′ end of the corresponding cDNA sequence. The tagsequence was then used to search the MPSS database again to reveal itsabundance in different tissues. As illustrated in Table 1, the PSO323364gene was confirmed to be flower-specific; the PSO400362 gene wasconfirmed to be seed-specific; and PSO332986 and PSO333268 wereconfirmed to be constitutively expressed. No sequence-specific tag wasidentified for PSO333209.

TABLE 1 Abundances of four gene-specific MPSS tags in soybean tissuesGene ID PSO323364 PSO400362 PSO332986 PSO333268 SEQ ID NO: 16 17 18 19Anther 0 0 200 2245 Flower 1720 0 3325 2715 Leaf 0 0 2105 4810 Pod 0 03327 5848 Root 0 0 6046 4422 Seed 0 82124 4338 7171 Stem 0 0 3827 3275

The MPSS profiles of the candidate genes were confirmed and extended byanalyzing 14 different soybean tissues using the relative quantitativeRT-PCR (qRT-PCR) technique with an ABI7500 real time PCR system (AppliedBiosystems, Foster City, Calif.).

Fourteen soybean tissues (somatic embryo, somatic embryo grown one weekon charcoal plate, leaf, leaf petiole, root, flower bud, open flower, R3pod, R4 seed, R4 pod coat, R5 seed, R5 pod coat, R6 seed, R6 pod coat)were collected from cultivar ‘Jack’ and flash frozen in liquid nitrogen.The seed and pod development stages were defined according todescriptions in Fehr and Caviness, IWSRBC 80:1-12 (1977). Total RNA wasextracted with Trizol reagents (Invitrogen, Carlsbad, Calif.) andtreated with DNase I to remove any trace amount of genomic DNAcontamination. The first strand cDNA was synthesized using theSuperscript III reverse transcriptase (Invitrogen).

PCR analysis was performed to confirm that the cDNA was free of genomicDNA, using primers SAMS-L and SAMS-L2 (SEQ ID NO:53 and SEQ ID NO:54,respectively). The primers are specific to the 5′UTR intron/exonjunction region of a soybean S-adenosylmethionine synthetase genepromoter SAMS (PCT Publication No. WO00/37662). PCR using this primerset amplifies a 967 bp DNA fragment from soybean genomic DNA templateand a 376 bp DNA fragment from the cDNA template.

The cDNA aliquots were used in the quantitative RT-PCR analysis, usingthe Power Sybr® Green real time PCR master mix (Applied Biosystems). Anendogenous soybean ATP sulfurylase gene was used as an internal control,and wild type soybean genomic DNA was used as the calibrator forrelative quantification. The data was captured and analyzed using thesequence detection software provided with the ABI7500 real time PCRsystem. The gene-specific primers used for the endogenous control ATPSgene were ATPS-87F and ATPS-161R (SEQ ID NO:20 and SEQ ID NO:21,respectively). The primers used for the five other target genes were:PSO323364F and PSO323364R (SEQ ID NO:22 and SEQ ID NO:23, respectively)for PSO323364, PSO400362F and PSO400362R (SEQ ID NO:24 and SEQ ID NO:25,respectively) for PSO400362, PSO332986F and PSO332986R (SEQ ID NO:26 andSEQ ID NO:27, respectively) for PSO332986, PSO333268F and PSO333268R(SEQ ID NO:28 and SEQ ID NO:29, respectively) for PSO333268, andPSO333209F and PSO333209R (SEQ ID NO:30 and SEQ ID NO:31, respectively)for PSO333209. For each of the five genes, the qRT-PCR profile, asillustrated in FIG. 1, was consistent with its respective MPSSexpression profile.

The putatively translated polypeptide sequences of the five candidategenes were used to search the databases of the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/) to identifyhomologous sequences. The putative polypeptide encoded by the PSO323364gene (the nucleotide and amino acid sequences are set forth in SEQ IDNO:6 and SEQ ID NO:11, respectively) has significant homology to MYBtranscription factors (Uimari and Strommer, Plant J. 12:1273-1284(1997)) and is referred to herein as MYB2. The putative polypeptideencoded by the PSO400362 gene (the nucleotide and amino acid sequencesof which are set forth in SEQ ID NO:7 and SEQ ID NO:12, respectively)has significant homology to Kunitz trypsin inhibitors (Jofuko andGoldberg, Plant Cell 1:1079-1093 (1989)) and is referred to herein asKTI1. The putative polypeptide encoded by the PSO332986 gene (thenucleotide and amino acid sequences of which are set forth in SEQ IDNO:8 and SEQ ID NO:13, respectively) has significant homology to plasmamembrane intrinsic proteins (Uehlein et al., Phytochemistry 68:122-129(2007)) and is referred to herein as PIP1. The putative polypeptideencoded by the PSO333268 gene (the nucleotide and amino acid sequencesof which are set forth in SEQ ID NO:9 and SEQ ID NO:14, respectively)has significant homology to translation elongation factor EF-1α genes(Aguilar et al., Plant Mol. Biol. 17:351-360 (1991)) and is referred toherein as EF1A2. The putative polypeptide encoded by the PSO333209 gene(the nucleotide and amino acid sequences of which are set forth in SEQID NO:10 and SEQ ID NO:15, respectively) has significant homology tometallothionein-like proteins (Munoz et al., Physiol. Plantarum104:273-279 (1998)) and is referred to herein as MTH1.

Example 2 Cloning of Novel Terminators

Soybean BAC (bacterial artificial chromosome) clones that contain theselected genes were identified by PCR analysis. PSO323364 was found onBAC clone SBH145N17; PSO400362 was found on BAC clone SBH136J24;PSO332986 was found on BAC clone SBH172F4; PSO333268 was found on BACclone SBH123F11; and PSO333209 was found on BAC clone SBH85K11.Approximately 1 kb of 3′ end sequence for each of the selected cDNAs wassequenced from each respective BAC clone, in order to amplify terminatorsequences via PCR. For each PCR, a SacI site (GAGCTC) was introduced bythe 5′ end sense primer and an EcoRI site (GAATTC) was introduced by the3′ antisense primer. Hence, primers PSO323364Sac and PSO323364Eco (SEQID NO:40 and SEQ ID NO:41, respectively) were used to amplify the MYB2terminator; primers PSO400362Sac and PSO400362Eco (SEQ ID NO:42 and SEQID NO:43, respectively) were used to amplify the KTI1 terminator;primers PSO332986Sac and PSO332986Eco (SEQ ID NO:44 and SEQ ID NO:45,respectively) were used to amplify the PIP1 terminator; primersPSO333268Sac and PSO333268Eco (SEQ ID NO:46 and SEQ ID NO:47,respectively) were used to amplify the EF1A2 terminator; and primersPSO333209Sac and PSO333209Eco (SEQ ID NO:48 and SEQ ID NO:49,respectively) were used to amplify the MTH1 terminator. PCR cycleconditions were 94° C. for 4 minutes; 35 cycles of 94° C. for 30seconds, 60° C. for 1 minute, and 68° C. for 1 minute; and a final 68°C. for 5 minutes before holding at 4° C. using the Platinum highfidelity Taq DNA polymerase (Invitrogen). PCR reactions were resolvedusing agarose gel electrophoresis to identify DNA bands representing the˜0.5 Kb terminators.

Each PCR amplified a terminator DNA fragment. The MYB2 terminatorfragment was digested with SacI and EcoRI and then ligated to thecorresponding SacI and EcoRI sites of Gateway entry vector QC315 (FIG.2A and SEQ ID NO:32), to create an intermediate construct QC327 (FIG. 2Band SEQ ID NO:33). Several clones of QC327 were sequenced, and the clonewith the correct MYB2 terminator sequence (SEQ ID NO:1) was selected. Inconstruct QC327, the MYB2 terminator was placed downstream of thefluorescent reporter gene ZS-YELLOW N1 (YFP), which was under thecontrol of a soybean ubiquitin promoter GM-UBQ. The YFP expressioncassette was then linked to a soybean transformation selectable markergene cassette SAMS:HRA in construct QC324i (FIG. 2C and SEQ ID NO:34) byLR clonase-mediated DNA recombination between the attL1 and attL2recombination sites (SEQ ID NO:87 and SEQ ID NO:88, respectively) inQC327 and the attR1 and attR2 recombination sites (SEQ ID NO:89 and SEQID NO:90, respectively) in QC324i (Invitrogen), to create the finaltransformation ready construct QC339 (FIG. 2D and SEQ ID NO:35). Two 21bp recombination sites attB1 and attB2 (SEQ ID NO:91 and SEQ ID NO:92,respectively) were created, resulting from DNA recombination betweenattL1 and attR2 and from DNA recombination between attL2 and attR2,respectively. Similarly, the other four terminators, KTI1 (SEQ ID NO:2),PIP1 (SEQ ID NO:3), EF1A2 (SEQ ID NO:4), and MTH1 (SEQ ID NO:5), werecloned into the final transformation ready constructs, QC340, QC350,QC351, and QC352, respectively (FIG. 3A, 3B, 3C, 3D). Complete sequencesof constructs QC339, QC340, QC350, QC351, and QC352 are listed as SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39,respectively.

Example 3 Transformation of Soybean with the Terminator Constructs

Each of the terminator constructs, QC339, QC340, QC350, QC351, andQC352, contained a DNA fragment that included the respectiveUBQ:YFP:terminator cassette linked to a SAMS:HRA expression cassette.For each construct, the DNA fragment was isolated by digestion withAscI, separated from the vector backbone by agarose gel electrophoresis,and gel-purified using a DNA gel extraction kit (Qiagen, Valencia,Calif.). Each of the purified DNA fragments was transformed into thesoybean cultivar “Jack” by particle gun bombardment (Klein et al.,Nature 327:70-73 (1987); U.S. Pat. No. 4,945,050), as described indetail below, to study the functions of each terminator in stablytransformed soybean plants.

The same methodology as outlined above for theUBQ:YFP:terminator-SAMS:HRA expression cassette construction andtransformation can be used with other heterologous nucleic acidsequences encoding for example a reporter protein, a selection marker, aprotein conferring disease resistance, a protein conferring herbicideresistance, a protein conferring insect resistance, a protein involvedin carbohydrate metabolism, a protein involved in fatty acid metabolism,a protein involved in amino acid metabolism, a protein involved in plantdevelopment, a protein involved in plant growth regulation, a proteininvolved in yield improvement, a protein involved in drought resistance,a protein involved in cold resistance, a protein involved in heatresistance, and a protein involved in salt resistance, all in plants.

Soybean somatic embryos from the Jack cultivar were induced as follows.Cotyledons (˜3 mm in length) were dissected from surface sterilized,immature seeds and were cultured for 6-10 weeks in the light at 26° C.on Murashige and Skoog (MS) media containing 0.7% agar and supplementedwith 10 mg/ml 2,4-D. Globular stage somatic embryos, which producedsecondary embryos, were excised, placed into flasks containing liquid MSmedium supplemented with 2,4-D (10 mg/ml), and cultured in the light ona rotary shaker. After repeated selection for clusters of somaticembryos that multiplied as early, globular staged embryos, the soybeanembryogenic suspension cultures were maintained in 35 ml liquid media ona rotary shaker, 150 rpm, at 26° C. with fluorescent lights on a 16:8hour day/night schedule. Cultures were subcultured every two weeks byinoculating approximately 35 mg of tissue into 35 ml of the same freshliquid MS medium.

Soybean embryogenic suspension cultures were then transformed by themethod of particle gun bombardment using a DuPont Biolistic™ PDS1000/HEinstrument (helium retrofit) (Bio-Rad Laboratories, Hercules, Calif.).To 50 μl of a 60 mg/ml 1.0 mm gold particle suspension were added (inorder): 30 μl of 10 ng/μl QC339 DNA fragment UBQ:YFP:MYB2-SAMS:HRA,QC340 DNA fragment UBQ:YFP:KTI1-SAMS-HRA, QC350 DNA fragmentUBQ:YFP:PIP1-SAMS-HRA, QC351 DNA fragment UBQ:YFP:EF1A2-SAMS-HRA, orQC352 DNA fragment UBQ:YFP:MTH1-SAMS-HRA; 20 μl of 0.1 M spermidine; and25 μl of 5 M CaCl₂. The particle preparation was then agitated for 3minutes and spun in a centrifuge for 10 seconds, and the supernatant wasremoved. The DNA-coated particles were then washed once in 400 μl 100%ethanol and resuspended in 45 μl of 100% ethanol. The DNA/particlesuspension was sonicated three times for one second each. 5 μl of theDNA-coated gold particles was then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture was placedin an empty 60×15 mm Petri dish, and the residual liquid removed fromthe tissue with a pipette. For each transformation experiment,approximately 5 to 10 plates of tissue were bombarded. Membrane rupturepressure was set at 1100 psi, and the chamber was evacuated to a vacuumof 28 inches mercury. The tissue was placed approximately 3.5 inchesaway from the retaining screen and bombarded once. Followingbombardment, the tissue was divided in half and placed back into liquidmedia and cultured as described above.

Five to seven days post bombardment, the liquid media was exchanged withfresh media containing 100 ng/ml chlorsulfuron, the selection agent. Theselective media was refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue was observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue wasremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each clonallypropagated culture was treated as an independent transformation eventand subcultured in the same liquid MS media supplemented with 2,4-D (10mg/ml) and 100 ng/ml chlorsulfuron selection agent to increase mass. Theembryogenic suspension cultures were then transferred to solid agar MSmedia plates without 2,4-D supplement to allow somatic embryos todevelop. A sample of each event was collected at this stage forquantitative PCR analysis.

Cotyledon stage somatic embryos were dried-down (by transferring theminto an empty small Petri dish that was seated on top of a 10 cm Petridish containing some agar gel to allow slow dry down) to mimic the laststages of soybean seed development. Dried-down embryos were placed ongermination solid media, and transgenic soybean plantlets wereregenerated. The transgenic plants were then transferred to soil andmaintained in growth chambers for seed production.

During the transformation, genomic DNA was extracted from somatic embryosamples and analyzed by quantitative PCR using the 7500 real time PCRsystem (Applied Biosystems) with gene-specific primers and FAM-labeledfluorescence probes to check copy numbers of both the UBQ:YFP:terminatorexpression cassette and the SAMS:HRA expression cassette. The qPCRanalysis was done in duplex reactions with a heat shock protein (HSP)gene as the endogenous control and a transgenic DNA sample with a knownsingle copy of SAMS:HRA or YFP transgene as the calibrator using therelative quantification methodology (Applied Biosystems). The endogenouscontrol HSP probe (SEQ ID NO:69) was labeled with VIC, and the YFP andSAMS probes (SEQ ID NO:66 and SEQ ID NO:63, respectively) were labeledwith FAM for the simultaneous detection of both fluorescent probes(Applied Biosystems). Gene cassette-specific primers used in the qPCRwere: SAMS48-F and SAMS-134R (SEQ ID NO:62 and SEQ ID NO:64,respectively) for SAMS:HRA, YFP-67F and YFP-130R (SEQ ID NO:65 and SEQID NO:67, respectively) for YFP, and HSP-F1 and HSP-R1 (SEQ ID NO:68 andSEQ ID NO:70, respectively) for the endogenous control gene HSP.

Only transgenic soybean events containing 1 or 2 copies of both theSAMS:HRA expression cassette and the UBQ:YFP expression cassette wereselected for further gene expression evaluation and seed production (seeTable 2). Events negative for YFP qPCR or with more than 2 copies forthe SAMS:HRA qPCR were not advanced. Four events from each terminatorconstruct were selected for RT-PCR analysis of gene expression, mRNApolyadenylation, and transcription termination, as described in EXAMPLES5, 6, and 7. YFP expressions are described in detail in EXAMPLE 4 andare also summarized in Table 2.

TABLE 2 Relative transgene copy numbers and YFP expression of terminatorconstructs in transgenic soybeans YFP SAMS Terminator Event ID YFP qPCRqPCR MYB2 4906.1.1 + 0.0 0.0 MYB2 4906.1.2 + 1.1 0.5 MYB2 4906.1.3 + 0.40.2 MYB2 4906.1.4 + 0.6 0.4 MYB2 4906.1.8 + 0.1 0.1 MYB2 4906.1.9 + 0.71.0 MYB2 4906.2.1 + 0.5 0.5 MYB2 4906.2.2 + 0.8 0.3 MYB2 4906.2.4 + 0.10.1 MYB2 4906.2.5 + 0.2 0.1 MYB2 4906.6.2 + 1.6 1.5 MYB2 4906.7.1 + 1.10.5 MYB2 4906.8.1 + 1.5 1.6 MYB2 4906.8.2 + 0.8 0.6 MYB2 4906.8.5 + 0.10.0 MYB2 4906.8.7 + 1.7 1.6 KTI1 4909.1.1 + 1.3 1.4 KTI1 4909.1.2 + 1.21.7 KTI1 4909.2.1 + 1.2 1.7 KTI1 4909.2.2 + 0.9 1.1 KTI1 4909.2.4 + 1.01.5 KTI1 4909.4.2 + 1.0 1.3 KTI1 4909.5.1 + 1.1 1.0 KTI1 4909.7.1 + 1.31.4 KTI1 4909.7.2 + 1.2 1.9 KTI1 4909.8.1 + 1.4 1.8 KTI1 4909.8.2 + 1.21.3 KTI1 4909.8.3 + 1.1 1.0 KTI1 4909.8.4 + 1.0 1.1 KTI1 4909.8.5 + 0.81.2 PIP1 4952.1.2 + 1.6 1.1 PIP1 4952.2.1 + 1.6 1.3 PIP1 4952.3.1 + 3.31.6 PIP1 4952.4.2 + 2.2 2.2 PIP1 4952.4.4 + 1.0 0.8 PIP1 4952.7.1 + 1.21.7 PIP1 4952.7.2 + 1.3 1.0 PIP1 4952.3.3 + 1.2 1.6 PIP1 4952.3.5 + 1.60.7 PIP1 4952.4.5 + 2.9 2.0 PIP1 4952.4.6 + 3.5 0.6 PIP1 4952.4.7 + 3.71.5 PIP1 4952.4.10 + 0.9 0.7 PIP1 4952.7.3 + 3.0 1.2 PIP1 4952.7.4 + 2.91.5 PIP1 4952.7.5 + 1.0 0.7 EF1A2 4953.1.2 + 1.0 1.0 EF1A2 4953.1.5 +1.1 0.6 EF1A2 4953.2.2 + 1.0 1.0 EF1A2 4953.4.7 + 1.0 1.4 EF1A24953.4.9 + 0.9 0.9 EF1A2 4953.5.3 + 1.0 0.7 EF1A2 4953.5.4 + 0.9 0.6EF1A2 4953.5.5 + 0.9 0.9 EF1A2 4953.5.6 + 0.9 1.4 EF1A2 4953.6.1 + 1.01.3 EF1A2 4953.6.2 + 0.8 0.9 EF1A2 4953.6.6 + 0.9 1.0 EF1A2 4953.6.10 +0.9 1.0 EF1A2 4953.7.1 + 0.9 0.7 EF1A2 4953.7.3 + 0.9 0.8 EF1A24953.8.1 + 1.1 1.0 EF1A2 4953.8.2 + 1.1 0.9 MTH1 5238.8.1 + 0.9 0.5 MTH15238.8.2 + 1.7 0.5 MTH1 5238.8.4 + 1.1 0.9 MTH1 5238.2.1 + 1.1 1.4 MTH15238.2.4 + 2.0 1.1 MTH1 5238.2.5 + 1.2 0.5 MTH1 5238.2.6 + 1.0 0.8 MTH15238.2.8 + 1.0 0.7 MTH1 5238.3.2 + 1.3 1.3 MTH1 5238.3.3 + 1.0 0.9 MTH15238.7.1 + 1.3 0.8 MTH1 5238.8.6 + 1.3 0.7 MTH1 5238.8.7 + 1.0 0.9 MTH15238.7.10 + 1.6 0.7 MTH1 5238.7.12 + 0.6 0.9 MTH1 5238.7.13 + 1.3 0.6

Example 4 YFP Expression in Stable Transgenic Soybeans

YFP gene expression was tested at different stages of transgenic plantdevelopment for yellow fluorescence emission under a Leica MZFLIIIstereo microscope equipped with appropriate fluorescent light filters.Yellow fluorescence was detected early on during somatic embryodevelopment and throughout all stages of transgenic plant development inall tissues tested (including somatic embryos, leaf, stem, root, flower,pod, and seed) for all five terminator constructs, QC339, QC340, QC350,QC351, and QC352. Since all five terminators were tested in the contextof the same soybean ubiquitin promoter GM-UBQ, the reporter expressionpatterns and strengths were similar among the five constructs suggestingthat the terminators did not change the overall expression of thepromoter. The five terminators could not be distinguished from eachother in terms of reporter gene expression. Examples of YFP expressionin flower, leaf, stem, root, and pod/seed are described below and shownin FIG. 4 (for the MYB2 terminator construct QC339, FIG. 4A-E; for theKTI1 terminator construct QC340, FIG. 4F-J; for the PIP1 terminatorconstruct QC350, FIG. 4K-O; for the EF1A2 terminator construct QC351,FIG. 4P-T; and for the MTH1 terminator construct QC352, FIG. 4U-Y).

During the tissue culture stages of transgenic plant regeneration, YFPexpression was detected in globular, torpedo, fully developed, and drieddown somatic embryos. Negative control embryos emitted weak red color,as did the negative sectors of positive embryo clusters, due toautofluorescence from the chlorophyll contained in green tissues,including somatic embryos. Negative controls for other green tissues,such as leaf or stem, were also red, and negative controls for whitetissues, such as root and flower petal, were dark yellowish under theYFP light filter. When transgenic plantlets were regenerated, YFPexpression was detected in leaf, stem, and root, and the expression wasretained to mature plants. Fluorescence in leaflets collected fromplantlets seemed stronger than in leaves collected from mature plants,probably due in part to the weak masking effect of less chlorophyll onyellow fluorescence in young leaves. Fluorescence was concentrated inthe vascular bundles in stems and roots. Strong yellow fluorescence wasunanimously detected in reproductive organs such as flowers anddeveloping pods and seeds, including seed coats and embryos, at allstages. In conclusion, each of the five novel terminators gaveconstitutive YFP expression under the control of the soybean ubiquitinpromoter.

Example 5 Gene Expression Evaluation by RT-PCR

To evaluate the functions of the terminators, total RNA was extractedfrom selected transgenic plantlets using the Trizol reagent followingthe protocol recommended by the manufacturer (Invitrogen). RNA sampleswere treated with RNase-free DNase I (Invitrogen) to get rid of anypotential genomic DNA contamination and checked by RT-PCR with primersSAMS-L (SEQ ID NO:53) and SAMS-L2 (SEQ ID NO:54), which are specific toan endogenous S-adenosylmethionine synthetase gene. Since the SAMS-Lprimer is specific to the upstream of a 5′UTR intron of the SAMS geneand the SAMS-L2 primer is specific to the coding region downstream ofthe same intron, any SAMS genomic DNA will produce a 967 bp PCR band,while the SAMS cDNA will produce a 376 bp RT-PCR band.

A typical 25 μl RT-PCR reaction was set up with 100 ng total RNA, 200 nMsense primer, 200 nM antisense primer, and 12.5 μl 2× one-step RT-PCRreaction mix (Invitrogen). The RT-PCR program included 30 minutes at 50°C. for the first strand cDNA synthesis; 3 minutes at 94° C. for theinitial denaturing; and 35 cycles of 30 seconds at 94° C., 1 minute at60° C., and 1 minute at 72° C. A final incubation at 72° C. for 5minutes was included before holding at 4° C. RT-PCR products wereresolved in 1% agarose gels by electrophoresis. All RNA samples werechecked by this assay and were determined to be genomic DNA-free asshown in FIG. 6A, where no 967 bp band, specific to genomic DNA, wasamplified from any of the RNA samples. Since the SAMS-L primer is at thefar 5′ end of the SAMS gene, the successful amplification of the 376 bpRT-PCR band from all the RNA samples also confirmed that each of the RNAsequences was full-length. A similar check was also done with primersUBQ-S2 (SEQ ID NO:61) and YFP-A (SEQ ID NO:60), which were specific tothe UBQ:YFP transgene, to further confirm that the RNAs were full-lengthand free of genomic DNA contamination.

In addition to the UBQ-S2/YFP-A primers used to check the transgenetranscripts, other transgene-specific primers were designed fordifferent analytical purposes (FIG. 5). To check the UBQ:YFP transgeneexpression, four independent events selected from each of the fiveterminator transformations were analyzed by RT-PCR, as described above,with primers YFP-1 (SEQ ID NO:57) and YFP-2 (SEQ ID NO:58). For eachterminator transformation, all four samples produced the expected YFPband with the same intensity, indicating that all of the testedtransgenic events expressed the YFP transgene similarly (FIG. 6B).

Example 6 Evaluation of Transgene Transcription Termination

The ability of the terminators to terminate RNA transcription wasanalyzed by RT-PCR using the sense primer YFP-3 (SEQ ID NO:59), which isspecific to the YFP gene, and an antisense primer specific to a regiondownstream from and beyond the terminator in the transgene construct.Since all five terminator constructs have the same configuration, thesame antisense primers, 3UTR-3 (SEQ ID NO:52), SAMS-A1 (SEQ ID NO:55),and SAMS-A2 (SEQ ID NO:56), could be used to analyze the progressivetermination of transcripts for all five terminators, MYB2, KTI1, PIP1,EF1A2, and MTH1 (FIG. 5). If RNA transcription was terminated 100% bythe terminator, no RT-PCR band would be detected with any of the primerset. If the transcription termination was not 100% effective and someRNA transcripts read through and beyond the terminator region, a band ofexpected size would be amplified by the RT-PCR with each respectiveprimer set.

Most of the transgenic events showed RNA transcription read throughbands with all three primer sets, YFP-3/3UTR-3, YFP-3/SAMS-A1, andYFP-3/SAMS-A2 (FIG. 7). The RT-PCR with primers YFP-3/3UTR-3 alsoamplified an approximately 400 bp non-specific band from wild type RNAin addition to the specific RT-PCR bands from the transgenic RNAtemplates (FIG. 7A). The larger transgene transcript-specific bands wereamplified from each of the four samples of all five terminators MYB2,KTI1, PIP1, EF1A2, and MTH1, except for the first MYB2 sample. Anintense smaller band was amplified for this sample suggesting that thetransgene probably contained a deletion in the terminator region in thisMYB2 event. Indeed, a 3′ UTR sequence recovered from this eventcontained a 362 bp deletion in the middle of the MYB2 terminator(EXAMPLE 8). A transgene read through-specific band slightly larger thanthe wild type non-specific band was also amplified from the fourtransgenic events carrying a control transgene operably linked to thepotato PIN2 terminator. Expected sizes of the transgene-specific bandsare given in FIG. 7 adjacent to the terminator names. The resultsindicated that none of the terminators, including the commonly usedPIN2, stopped RNA transcription completely. Instead, transcriptionread-through beyond the 3′ end of the terminators occurred frequently,though at various levels, as indicated by the different intensities ofthe bands.

Since the 3UTR-3 antisense primer is only 38 bp (including the 23 bp3UTR-3 primer sequence) from the EcoRI site at the 3′ end of all theterminators, the extent of transcription read-through could be furtherevaluated using two more sets of primers further downstream of theterminator. The antisense primer SAMS-A1 used in the second RT-PCR is248 bp downstream of the 3UTR-3 primer used in the first RT-PCR, or 286bp downstream of the 3′ end (the EcoRI cloning site) of all theterminators. The antisense primer SAMS-A2 used in the third RT-PCR is174 bp downstream of the SAMS-A1 primer, or 422 bp downstream of the3UTR-3 primer, or 460 bp downstream of the terminator's 3′ end. Thesecond set of primers, YFP-3/SAMS-A1, amplified RT-PCR bands from allthe KTI1, PIP1, and EF1A2 samples, although the second EF1A2 sample hada very faint band. The number 4 MYB2 sample, the number 2 MTH1 sample,and all four PIN2 samples produced faint bands of the expected sizes.The first MYB2 samples again produced an intense but smaller band. (FIG.7B). The third set of primers, YFP-3/SAMS-A2, amplified similar bands,although there were slight changes in intensity for some of the bands,as compared to the second RT-PCR (FIG. 7C). Some bands became weaker andsome bands became stronger, presumably due to PCR variations. Theresults indicated that RNA transcription did not terminate at one site,with some RNA transcripts extending as far as 460 bp or longerdownstream of the 3′ end of the tested terminator. Transcriptionread-through was less severe in terminators MYB2, MTH1, and PIN2, ascompared to terminators PIP1, KTI1, and EF1A2.

To check the percentage of transcription read through, the relativequantity of the YFP transcripts estimated by qRT-PCR was compared tothat of the SAMS promoter (FIG. 5). The sense primer YFP-139F (SEQ IDNO:74), probe YFP-160T (SEQ ID NO:75), and antisense primer YFP-195R(SEQ ID NO:76), all of which are specific to the 3′ end of the YFPcoding region, were used for YFP-specific qRT-PCR. The qRT-PCR specificto the SAMS promoter designed to locate further downstream of theterminators used sense primer SamsPro-F (SEQ ID NO:71), probe SamsPro-T(SEQ ID NO:72), and antisense primer SamsPro-R (SEQ ID NO:73). Theantisense primer SamsPro-R is 553 bp downstream of the 3UTR-3 primer or591 bp downstream of the 3′ end EcoRI site of the terminators. Theendogenous ATP sulfurylase gene, detected with sense primer ATPS-87F(SEQ ID NO:20), probe ATPS-117T (SEQ ID NO:93), and antisense primerATPS-161R (SEQ ID NO:21), was used as the endogenous control for boththe YFP and SAMS promoter qRT-PCR. The genomic DNA of a transgenicsoybean event containing one copy of both YFP and SAMS promoter was usedas a calibrator. The relative quantification (RQ) of YFP or SAMSpromoter was calculated using the 7500 system SDS software (AppliedBiosystems). RNA transcription read through frequency was expressed asthe percentage of the expression of SAMS promoter to that of YFP foreach tested sample. Four independent transgenic events were analyzed foreach of the five terminators. Transcription read through was detected inall the events though most of them were less than 1% as listed in thelast column of Table 3.

TABLE 3 Relative quantification of RNA transcription read through byqRT-PCR RNA SamsPro- SamsPro/ Terminator source Event YFP-RQ RQ YFP MYB2Embryo 4906.1.2 0.188 0.008 4.26% Embryo 4906.5.3 1.13 0.005 0.44%Embryo 4906.6.1 0.566 1.94E−04 0.03% Embryo 4906.8.1 1.144 2.79E−040.02% KTI1 Leaf 4909.2.4.2 11.05 0.037 0.33% Leaf 4909.7.1.2 5.25 0.0250.48% Leaf 4909.8.2.2 7.166 1.31 18.28% Leaf 4909.8.3.1 7.499 0.0290.39% PIP1 Plantlet 4952.3.1 19.873 0.081 0.41% Plantlet 4952.4.4 6.5110.001 0.02% Plantlet 4952.4.10 4.881 0.02 0.41% Plantlet 4952.7.3 15.5280.038 0.24% EF1A2 Embryo 4953.1.1 0.452 0.003 0.66% Embryo 4953.2.10.009 2.59E−04 2.88% Embryo 4953.4.4 0.757 0.007 0.92% Embryo 4953.5.10.685 0.012 1.75% MTH1 Plantlet 5238.2.12 25.548 0.018 0.07% Plantlet5238.6.8 13.572 0.025 0.18% Plantlet 5238.7.11 9.022 0.023 0.25%Plantlet 5238.7.12 6.943 0.009 0.13%

Example 7 Evaluation of Endogenous Gene Transcription Termination

Though it is believed that transcription termination by bacterial RNApolymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpinfollowed by a U-rich tract (Gusarov and Nudler, Mol. Cell 3:495-504(1999), Larson et al., Cell 132:971-982 (2008)), little is known abouttranscription termination in plants. To check if transcription readthrough observed in transgenes is also common for endogenous genes,primers were designed to check RNA transcripts of each of the fiveendogenous genes corresponding to the five terminators. The first set ofprimers, specific to normal mature mRNA, consisted of a sense primerspecific to the coding region and an antisense primer specific to the3UTR upstream of the poly (A). An RT-PCR band of a specific size wouldbe expected from soybean wild type total RNA and from genomic DNApositive control. The second set of primers, specific to read throughtranscripts or precursor mRNA, consisted of the same sense primerspecific to the coding region and an antisense primer specific to aregion approximately 100-300 bp downstream of the poly (A). Iftranscription read through did occur, a larger band would be expectedfrom total RNA by RT-PCR and from genomic DNA by PCR. If transcriptionread through did not occur, only the genomic DNA templates would producethe larger PCR band. The RNA templates would not produce the larger bandif RNA transcripts did not extend 100-300 bp beyond the polyadenylationsite.

RT-PCR analysis was done on wild type RNA extracted from soybeanplantlets for each of the five terminators MYB2, KTI1, PIP1, EF1A2, andMTH1 (FIG. 8A). The RNA used in the assays had been checked to be freeof any genomic DNA contamination and the band detected in the RNAsamples had to come from RNA by RT-PCR. Genomic DNA was included aspositive control, and water was used as a no template control for eachset of primers. The RT-PCR-1 used normal mRNA-specific primer sets, andthe RT-PCR-2 used transcription read-through specific primer sets (FIG.8A). The normal mRNA-specific primer sets used in RT-PCR-1 were:PSO323364S1/PSO323364R1 (SEQ ID NO:77/SEQ ID NO:78),PSO400362S1/PSO400362R1 (SEQ ID NO:79/SEQ ID NO:80),PSO332982F/PSO332986JK-A (SEQ ID NO:81/SEQ ID NO:82),PSO333268F/PSO333268R (SEQ ID NO:83/SEQ ID NO:84), andPSO333209F/PSO333209JK-A (SEQ ID NO:85/SEQ ID NO:86), respectively, forthe MYB2, KTI1, PIP1, EF1A2, and MTH1 terminators. The transcriptionread-through specific primer sets used in RT-PCR-2 werePSO32336451/PSO323364Eco (SEQ ID NO:77/SEQ ID NO:41),PSO40036251/PSO400362Eco (SEQ ID NO:79/SEQ ID NO:43),PSO332982F/PSO332986Eco (SEQ ID NO:81/SEQ ID NO:45),PSO333268F/PSO333268Eco (SEQ ID NO:83/SEQ ID NO:47), andPSO333209F/PSO333209Eco (SEQ ID NO:85/SEQ ID NO:49), respectively, forthe MYB2, KTI1, PIP1, EF1A2, and MTH1 terminators.

RT-PCR bands were detected with both the mRNA specific RT-PCR-1 and thetranscription read-through specific RT-PCR-2 from the RNA samples forPSO333209 (MTH1), PSO333268 (EF1A2), and PSO332986 (PIP1). As expected,the RT-PCR-2 bands are larger than the corresponding RT-PCR-1 bands.Bands of the same sizes were detected in the genomic DNA positivecontrols for all the primer sets (FIG. 8A). Since PSO323364 (MYB2) is aflower-specific gene and PSO400362 (KTI1) is an embryo-specific gene, noRT-PCR band was amplified with RT-PCR-1 or RT-PCR-2 from the plantletRNA samples for these two genes, while both amplified specific PCR bandsfrom the genomic DNA positive controls (FIG. 8A). Flower RNA and embryoRNA had to be used accordingly in order to check transcriptionread-through for these two tissue-specific genes (FIG. 8B). RT-PCR bandswere amplified by both RT-PCR-1 and RT-PCR-2 for PSO323364 (MYB2) fromthe flower RNA but not from the seed RNA, while for gene PSO400362(KTI1), from the seed RNA but not from the flower RNA (FIG. 8B). Boththe flower and seed RNA were also checked by RT-PCR with primersSAMS-L/SAMS-L2 (SEQ ID NO:53/SEQ ID NO:54) to be free of genomic DNAcontamination (FIG. 8B). Since the antisense primer used in eachRT-PCR-2 was 222 bp, 296 bp, 194 bp, 87 bp, and 195 bp from thepolyadenylation site (see SEQ ID NO:6, 7, 8, 9, and 10), respectively,for genes PSO323364 (MYB2), PSO400362 (KTI1), PSO332986 (PIP1),PSO333268 (EF1A2), and PSO333209 (MTH1), the results confirmed that RNAtranscription did not terminate at the corresponding position downstreamof the polyadenylation site for each of the five endogenous genes. Theterminators behaved similarly in their naturally endogenous genes aswhen they were in transgenic genes described in EXAMPLE 6.

Example 8 Cloning and Sequencing the 3′ UTRs of Transgenes

Transgenic 3′ UTRs were cloned by RT-PCR from the same four events foreach of the MYB2, KTI1, PIP1, EF1A2, and MTH1 terminators. First strandcDNA was made from each RNA sample with SuperScript III reversetranscriptase (Invitrogen), using the oligo dT primer 3UTR-1 (SEQ IDNO:50). The 3′ UTR, plus the 3′ part of the YFP coding region of eachtransgene, was amplified by PCR with primer set YFP-3/3UTR-2 (SEQ IDNO:59/SEQ ID NO:51). A single band was amplified for all but the fourMYB2 samples and one EF1A2 sample (FIG. 9). The PCR bands were thencloned into TOPO pCR2.1 vector by TA cloning (Invitrogen). Plasmid DNAwas obtained from each clone, using Qiagen plasmid mini kits, and theDNA was sequenced with M13For and M13Rev primers specific to the TOPOpCR2.1 vector. Sequences were analyzed using the ContigExpress andAlignX programs in Vector NTI suites (Invitrogen).

As summarized in Table 4, 19 specific 3′ UTR sequences representing 5different variants were recovered from the MYB2 events. The lengths ofthe five variants, starting from the 5′ SacI site of the MYB2terminator, are 143 bp, 198 bp, 244 bp, 341 bp, and 348 bp. In total,there were four 143 bp sequences, ten 198 bp sequences, one 244 bpsequence, two 341 bp sequences, and two 348 bp sequences. Two identical3′ UTR sequences (not listed in the table) cloned from the first MYB2event had the middle 362 bp of the MYB2 terminator deleted and thepolyadenylation site was outside the terminator, i.e., 43 bp downstreamof the 3UTR-3 primer or 81 bp downstream of the 3′ end (the EcoRI site)of the terminator. The observation is consistent with the smaller PCRbands amplified for the first MYB2 event (FIG. 7A, B, C). The middledeletion in the terminator made RNA transcription read through moresevere in this event than in the others since this event gave muchstronger bands for all three sets of primers used for the transcriptionread through check, as described in EXAMPLE 6.

Thirteen 3′ UTR sequences representing 5 variants were recovered fromthe KTI1 events; eleven 3′ UTR sequences representing only 2 variantswere recovered from the PIP1 events; twelve sequences representing 2variants were recovered from the EF1A2 events, and twenty five sequencesrepresenting 13 variants were recovered from the MTH1 events. It wasobvious that each terminator could have multiple polyadenylation sites.Since only limited numbers of clones were sequenced for each terminator,it was reasonable to believe that more polyadenylation sites could beidentified, especially for the MTH1, KTI1, and MYB2 terminators, sincesome of their 3′ UTR variants were represented by only single sequencesin Table 4.

TABLE 4 Summary of transgenic 3′ UTR sequence analysis Terminator MYB2KTI1 PIP1 EF1A2 MTH1 Gene PSO323364 PSO400362 PSO332986 PSO333268PSO333209 Native 3′ 305 243 309 345 259 UTR Construct QC339 QC340 QC350QC351 QC352 Full length 540 554 518 445 462 Transgene  19  13  11  12 25 3′ UTR sequence Transgene  5  5  2  2  13 3′ UTR variants Transgene143(4), 169(1), 319(7), 351(10), 213(1), 3′ UTR 198(10), 179(1), 337(4)369(2) 219(1), variants 244(1), 202(6), 240(1), lengths and 341(2),242(3), 259(8), frequency 348(2) 250(2), 263(1), 275(1), 277(1), 298(3),318(1), 324(3), 329(1), 343(2), 367(1)

Example 9 Identification and Cloning of Longer Versions of theTerminators

As described in EXAMPLE 7, transcription read through was detected inall five endogenous genes, PSO323364, PSO400362, PSO332986, PSO333268,and PSO333209, corresponding to terminators MYB2, KTI1, PIP1, EF1A2, andMTH1, respectively (FIG. 8). To check if the observed transcription readthrough of endogenous genes would stop and at what point, six moreprogressively downstream reverse primers were designed for each of thefive endogenous genes based on their genomic DNA sequences to do moreRT-PCR analyses. The relative positions of the single forward primerPSO323364S1 (SEQ ID NO:77) and seven reverse primers PSO323364Eco (SEQID NO:41), PSO323364UTR2 (SEQ ID NO:94), PSO323364UTR3 (SEQ ID NO:95),PSO323364UTR4 (SEQ ID NO:96), PSO323364UTR5 (SEQ ID NO:97),PSO323364UTR6 (SEQ ID NO:98), and PSO323364UTR7 (SEQ ID NO:99) specificto the RNA transcript and the genomic DNA of gene PSO323364 areillustrated in FIG. 10 as an example. RT-PCR analyses using the aboveseven sets of primers were labeled in the same order, as RT-PCR 1,RT-PCR 2, RT-PCR 3, RT-PCR 4, RT-PCR 5, RT-PCR 6, and RT-PCR 7 in FIG.11.

Similarly, primers were designed and RT-PCR was performed for the otherfour endogenous genes, PSO400362, PSO332986, PSO333268, and PSO333209.Forward primer PSO400362S1 (SEQ ID NO:79) and seven reverse primersPSO400362Eco (SEQ ID NO:43), PSO400362UTR2 (SEQ ID NO:100),PSO400362UTR3 (SEQ ID NO:101), PSO400362UTR4 (SEQ ID NO:102),PSO400362UTR5 (SEQ ID NO:103), PSO400362UTR6 (SEQ ID NO:104), andPSO400362UTR7 (SEQ ID NO:105) were used for the seven PSO400362-specificRT-PCR analyses (FIG. 11). Forward primer PSO332982F (SEQ ID NO:81) andseven reverse primers PSO332986Eco (SEQ ID NO:45), PSO332986UTR2 (SEQ IDNO:106), PSO332986UTR3 (SEQ ID NO:107), PSO332986UTR4 (SEQ ID NO:108),PSO332986UTR5 (SEQ ID NO:109), PSO332986UTR6 (SEQ ID NO:110), andPSO332986UTR7 (SEQ ID NO:111) were used for the seven PSO332986-specificRT-PCR analyses (FIG. 11). Forward primer PSO333268F (SEQ ID NO:83) andseven reverse primers PSO333268Eco (SEQ ID NO:47), PSO333268UTR2 (SEQ IDNO:112), PSO333268UTR3 (SEQ ID NO:113), PSO333268UTR4 (SEQ ID NO:114),PSO333268UTR5 (SEQ ID NO:115), PSO333268UTR6 (SEQ ID NO:116), andPSO333268UTR7 (SEQ ID NO:117) were used for the seven PSO333268-specificRT-PCR analyses (FIG. 11). Forward primer PSO333209F (SEQ ID NO:85) andseven reverse primers PSO333209Eco (SEQ ID NO:49), PSO333209UTR2 (SEQ IDNO:118), PSO333209UTR3 (SEQ ID NO:119), PSO333209UTR4 (SEQ ID NO:120),PSO333209UTR5 (SEQ ID NO:121), PSO333209UTR6 (SEQ ID NO:122), andPSO333209UTR7 (SEQ ID NO:123) were used for the seven PSO333209-specificRT-PCR analyses (FIG. 11).

RT-PCR analyses were done on the same wild type soybean flower RNA forgene PSO322264 (MYB2), seed RNA for PSO400362 (KTI1), or plantlet RNAfor genes PSO332986 (PIP1), PSO333268 (EF1A2), and PSO333209 (MTH1), asdescribed in EXAMPLE 7. The RNA used in the assays had been checked tobe free of any genomic DNA contamination and the band detected in theRNA samples had to come from RNA by RT-PCR. Genomic DNA was included aspositive control, and water was used as a no template control for eachset of primers. The genomic DNA positive would always give a band aslong as the RT-PCR worked. The RNA template would only give the samesize band only when there was transcription read through downstreambeyond the position of the reverse primer. If transcription read throughstopped, only the genomic DNA templates would produce the predicted PCRband. The same size band was detected in both the RNA and genomic DNAtemplate for each of the five endogenous genes until RT-PCR5 (FIG. 11A,B). RT-PCR 5 reactions were repeated to normalize the RT-PCR reactionsin FIG. 11A and in FIG. 11B that were done at different times. Probablydue to limited specific targets in the RNA templates, RT-PCR 5 resultswere not completely consistent between the corresponding reactions inFIG. 11A and in FIG. 11B for genes PIP1 and EF1A2. No specific band wasdetected in RT-PCR5 for MTH1 gene while a non-specific band was detectedin the same reaction (FIG. 11A). Non-specific bands of different sizeswere also detected in RT-PCR 6 for MYB2, PIP1, and EF1A2 genes. Theprimers failed for PIP1 gene RT-PCR 7 since no band was amplified eitherfrom the RNA template or the genomic DNA template (FIG. 11B). No RT-PCRband was amplified in RT-PCR 6 or RT-PCR 7 for any of the five genesindicating that transcription read through did not occur beyond thesixth reverse primer position.

The longer versions of the five terminators were amplified by PCR fromwild type soybean “Jack” genomic DNA using the same forward primersdescribed in EXAMPLE 2 and the UTR6 reverse primers. The MYB2Lterminator (SEQ ID NO: 124) was amplified with primers PSO323364Sac (SEQID NO:40) and PSO323364UTR6 (SEQ ID NO:98). The KTI1L terminator (SEQ IDNO:125) was amplified with primers PSO400362Sac (SEQ ID NO:42) andPSO400362UTR6 (SEQ ID NO:104). The PIP1L terminator (SEQ ID NO:126) wasamplified with primers PSO332986Sac (SEQ ID NO:44) and PSO332986UTR6(SEQ ID NO:110). The EF1A2L terminator (SEQ ID NO:127) was amplifiedwith primers PSO333268Sac (SEQ ID NO:46) and PSO333268UTR6 (SEQ IDNO:116). The MTH1L terminator (SEQ ID NO:128) was amplified with primersPSO333209Sac (SEQ ID NO:48) and PSO333209UTR6 (SEQ ID NO:122). PCR cycleconditions were 94° C. for 4 minutes; 35 cycles of 94° C. for 30seconds, 60° C. for 1 minute, and 68° C. for 2 minutes; and a final 68°C. for 5 minutes before holding at 4° C. using the Platinum highfidelity Taq DNA polymerase (Invitrogen). PCR reactions were resolvedusing agarose gel electrophoresis to identify DNA bands representing theapproximately 1.5 Kb terminators. Each longer terminator was then clonedin TOPO TA cloning vector pCR2.1-TOPO (Invitrogen) and confirmed bysequencing multiple clones. The longer terminators are used inconstructing transgenic gene cassettes wherever transcription readthrough needs to be limited.

What is claimed is:
 1. A recombinant DNA construct comprising apromoter, at least one heterologous nucleotide sequence, and an isolatedpolynucleotide comprising: a) a nucleotide sequence comprising thesequence set forth in SEQ ID NO:3; b) a nucleotide sequence comprising asequence having at least 95% sequence identity, based on the BLASTNmethod of alignment, when compared to the nucleotide sequence of (a); orc) the nucleotide sequence complementary to (a) or (b); wherein saidnucleotide sequence functions as a terminator and wherein the promoter,heterologous nucleotide sequence, and isolated polynucleotide areoperably linked.
 2. A recombinant DNA construct comprising a promoter,at least one heterologous nucleotide sequence, and an isolatedpolynucleotide comprising: a) a nucleotide sequence comprising afragment of SEQ ID NO:3; b) a nucleotide sequence comprising a sequencehaving at least 95% sequence identity, based on the BLASTN method ofalignment, when compared to the nucleotide sequence of (a); or c) thenucleotide sequence complementary to (a) or (b); wherein said nucleotidesequence functions as a terminator and wherein the promoter,heterologous nucleotide sequence, and isolated polynucleotide areoperably linked.
 3. A vector comprising the recombinant DNA construct ofclaim 1 or
 2. 4. A cell comprising the recombinant DNA construct ofclaim 1 or
 2. 5. The cell of claim 4, wherein the cell is a plant cell.6. A transgenic plant having stably incorporated into its genome therecombinant DNA construct of claim 1 or
 2. 7. The transgenic plant ofclaim 6 wherein said plant is a dicot.
 8. The transgenic plant of claim7 wherein said plant is soybean.
 9. Transgenic seed produced by thetransgenic plant of claim
 7. 10. A method of expressing a codingsequence or a functional RNA in a plant comprising: a) introducing therecombinant DNA construct of claim 1 or 2 into the plant, wherein the atleast one heterologous nucleotide sequence comprises the coding sequenceor the functional RNA; b) growing the plant of step a); and c) selectingthe plant displaying expression of the coding sequence or the functionalRNA of the recombinant DNA construct.
 11. A method of transgenicallyaltering a marketable plant trait, comprising: a) introducing therecombinant DNA construct of claim 1 or 2 into a plant cell; b) growinga fertile, mature plant from the plant cell resulting from step a); andc) selecting the plant expressing the at least one heterologousnucleotide sequence in at least one plant tissue based on the alteredmarketable trait.
 12. The method of claim 11 wherein the marketableplant trait is selected from the group consisting of: diseaseresistance, herbicide resistance, insect resistance, carbohydratemetabolism, fatty acid metabolism, amino acid metabolism, plantdevelopment, plant growth regulation, yield improvement, droughtresistance, cold resistance, heat resistance, and salt resistance.